Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase Pathway Research Articles

SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline†.

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.

Overcoming Steroid Resistance in Pediatric Acute Lymphoblastic Leukemia-The State-of-the-Art Knowledge and Future Prospects.

Acute lymphoblastic leukemia (ALL) is the most common malignancy among children. Despite the enormous progress in ALL therapy, resulting in achieving a 5-year survival rate of up to 90%, the ambitious goal of reaching a 100% survival rate is still being pursued. A typical ALL treatment includes three phases: remission induction and consolidation and maintenance, preceded by a prednisone prephase. Poor prednisone response (PPR) is defined as the presence of ≥1.0 × 109 blasts/L in the peripheral blood on day eight of therapy and results in significantly frequent relapses and worse outcomes. Hence, identifying risk factors of steroid resistance and finding methods of overcoming that resistance may significantly improve patients’ outcomes. A mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK-ERK) pathway seems to be a particularly attractive target, as its activation leads to steroid resistance via a phosphorylating Bcl-2-interacting mediator of cell death (BIM), which is crucial in the steroid-induced cell death. Several mutations causing activation of MAPK-ERK were discovered, notably the interleukin-7 receptor (IL-7R) pathway mutations in T-cell ALL and rat sarcoma virus (Ras) pathway mutations in precursor B-cell ALL. MAPK-ERK pathway inhibitors were demonstrated to enhance the results of dexamethasone therapy in preclinical ALL studies. This report summarizes steroids’ mechanism of action, resistance to treatment, and prospects of steroids therapy in pediatric ALL.

From the low-density lipoprotein receptor–related protein 1 to neuropathic pain: a potentially novel target

This review describes the roles of the low-density lipoprotein receptor-related protein 1 (LRP-1) in inflammatory pathways, nerve nerve degeneration and -regeneration and in neuropathic pain. Induction of LRP-1 is able to reduce the activation of the proinflammatory NFκB-mediated pathway and the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase and p38 signaling pathways, in turn decreasing the production of inflammatory mediators. Low-density lipoprotein receptor-related protein 1 activation also decreases reactive astrogliosis and polarizes microglial cells and macrophages from a proinflammatory phenotype (M1) to an anti-inflammatory phenotype (M2), attenuating the neuroinflammatory environment. Low-density lipoprotein receptor-related protein 1 can also modulate the permeability of the blood-brain barrier and the blood-nerve barrier, thus regulating the infiltration of systemic insults and cells into the central and the peripheral nervous system, respectively. Furthermore, LRP-1 is involved in the maturation of oligodendrocytes and in the activation, migration, and repair phenotype of Schwann cells, therefore suggesting a major role in restoring the myelin sheaths upon injury. Low-density lipoprotein receptor-related protein 1 activation can indirectly decrease neurodegeneration and neuropathic pain by attenuation of the inflammatory environment. Moreover, LRP-1 agonists can directly promote neural cell survival and neurite sprouting, decrease cell death, and attenuate pain and neurological disorders by the inhibition of MAPK c-Jun N-terminal kinase and p38-pathway and activation of MAPK extracellular signal-regulated kinase pathway. In addition, activation of LRP-1 resulted in better outcomes for neuropathies such as Alzheimer disease, nerve injury, or diabetic peripheral neuropathy, attenuating neuropathic pain and improving cognitive functions. To summarize, LRP-1 plays an important role in the development of different experimental diseases of the nervous system, and it is emerging as a very interesting therapeutic target.

The Synthetic Curcumin Derivative CNB-001 Attenuates Thrombin-Stimulated Microglial Inflammation by Inhibiting the ERK and p38 MAPK Pathways.

We have recently found that the synthetic curcumin derivative CNB-001 suppresses lipopolysaccharide (LPS)-induced nitric oxide (NO) production in cultured microglia, demonstrating that it exerts anti-neuroinflammatory effects by regulating microglial activation. To explore the molecular mechanisms underlying the anti-inflammatory effect of CNB-001, the present study investigated whether CNB-001 is also effective for microglial NO production induced by other stimulants than LPS. Treatment of primary cultured rat microglia with thrombin, a serine protease that has been proposed as a mediator of cerebrovascular injuries, caused the expression of inducible NO synthase (iNOS) and the production of NO. The thrombin-induced NO production was completely blocked by the presence of SCH-79797, a selective protease-activated receptor 1 (PAR-1) antagonist, suggesting that the effect of thrombin is mediated by PAR-1. CNB-001 (1-10 µM) attenuated the thrombin-induced iNOS expression and NO production without affecting the PAR-1 expression. In addition, thrombin treatment caused rapid phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). The changes in ERK and p38 MAPK were significantly suppressed by the presence of CNB-001. These results demonstrate that CNB-001 suppresses thrombin-stimulated microglial activation by inhibiting the ERK and p38 MAPK pathways.

Protective effects of probucol on Ox-LDL-induced epithelial-mesenchymal transition in human renal proximal tubular epithelial cells via LOX‑1/ROS/MAPK signaling.

Oxidized low-density lipoprotein (Ox-LDL), as a strong oxidant, results in renal injury through multiple mechanisms. The aim of the present study was to determine the injury effects of Ox‑LDL and the potential protective effects of the antioxidant reagent probucol on epithelial‑mesenchymal transition (EMT) in human renal proximal tubular epithelial cells (HK‑2) and to further explore the role and interrelation of lectin‑like oxidized low‑density lipoprotein receptor‑1 (LOX‑1), reactive oxygen species (ROS) and mitogen‑activated protein kinase (MAPK) pathway. In the present study, concentrations of 0‑100µg/ml Ox‑LDL were used to induce HK‑2 cell EMT. Then, probucol (20µmol/l) and the LOX‑1 inhibitor, polyinosinic acid (250µg/ml), were also used to pretreat HK‑2 cells. Intracellular ROS activity was evaluated using the specific probe 2',7'‑dichlorodihydrofluorescein diacetate (DCFH‑DA). Concentration of nitric oxide (NO) was determined using a biochemical colorimetric method. Expression of E‑cadherin, α‑smooth muscle actin (SMA), LOX‑1, NADPH oxidase 4 (NOX4), cytochrome b‑245 α chain (p22phox), extracellular signal‑regulated kinase (ERK), and p38 MAPK protein levels were examined by western blotting. The results revealed that Ox‑LDL induced the expression of LOX‑1 and α‑SMA and reduced the expression of E‑cadherin in a dose‑dependent manner, and these effects were inhibited by polyinosinic acid or probucol pretreatment. Stimulation with 50µg/ml Ox‑LDL induced the expression of NOX4 and p22phox and increased intracellular ROS activity, but NO production in the cell supernatants was not affected. The Ox‑LDL‑mediated increases in Nox4 and p22phox expression and in ROS activity were inhibited by probucol pretreatment. Further investigations into the underlying molecular pathways demonstrated that ERK and p38 MAPK were activated by Ox‑LDL stimulation and then inhibited by probucol pretreatment. The findings of the present study therefore suggest that Ox‑LDL induced EMT in HK‑2 cells, the mechanism of which may be associated with LOX‑1‑related oxidative stress via the ERK and p38 MAPK pathways. Notably, pretreatment with probucol inhibited the Ox‑LDL‑induced oxidative stress by reducing the expression of LOX‑1, and blocked the progression of EMT.

Spatial and temporal signal processing and decision making by MAPK pathways.

Mitogen-activated protein kinase (MAPK) pathways are conserved from yeast to man and regulate a variety of cellular processes, including proliferation and differentiation. Recent developments show how MAPK pathways perform exquisite spatial and temporal signal processing and underscores the importance of studying the dynamics of signaling pathways to understand their physiological response. The importance of dynamic mechanisms that process input signals into graded downstream responses has been demonstrated in the pheromone-induced and osmotic stress-induced MAPK pathways in yeast and in the mammalian extracellular signal-regulated kinase MAPK pathway. Particularly, recent studies in the yeast pheromone response have shown how positive feedback generates switches, negative feedback enables gradient detection, and coherent feedforward regulation underlies cellular memory. More generally, a new wave of quantitative single-cell studies has begun to elucidate how signaling dynamics determine cell physiology and represents a paradigm shift from descriptive to predictive biology.

Icariin induces osteogenic differentiation of bone mesenchymal stem cells in a MAPK-dependent manner.

Icariin, a flavonoid isolated from Epimedium pubescens, has previously been identified to exert beneficial effects on preventing bone loss and promoting bone regeneration. However, molecular mechanisms for its anabolic action have, up to now, remained largely unknown. Effects of icariin on cell proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) were systematically evaluated. To characterize underlying mechanisms, its effects on mitogen-activated protein kinase (MAPK) signalling pathways were determined. Results showed that icariin might not have enhanced effects on cell proliferation. However, it seemed to significantly enhance osteogenic differentiation of BMSCs, demonstrated by increasing alkaline phosphatase (ALP) activity and gene expression of collagen type I (Col I), osteocalcin (OCN) and osteopotin (OPN). It was demonstrated that icariin rapidly phosphorylated extracellular signal-regulated kinase (ERK), p38 kinase and c-Jun N terminal kinase (JNK). Furthermore, icariin-stimulated osteogenic effects on BMSCs were dramatically attenuated by treatment with either specific ERK inhibitor of PD98059, p38 inhibitor of SB202190 or JNK inhibitor SP600125. These results provide a potential mechanism of anabolic activity of icariin on BMSCs involving ERK, p38 and JNK MAPK pathways.

Metabolic respiration induces AMPK- and Ire1p-dependent activation of the p38-Type HOG MAPK pathway.

Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other's activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways.

TNF induces the expression of the sialyltransferase ST3Gal IV in human bronchial mucosa via MSK1/2 protein kinases and increases FliD/sialyl-Lewisx-mediated adhesion of Pseudomonas aeruginosa

We have shown previously that the pro-inflammatory cytokine TNF (tumour necrosis factor) could drive sLe(x) (sialyl-Lewis(x)) biosynthesis through the up-regulation of the BX transcript isoform of the ST3GAL4 (ST3 β-galactoside α-2,3-sialyltransferase 4) sialyltransferase gene in lung epithelial cells and human bronchial mucosa. In the present study, we show that the TNF-induced up-regulation of the ST3GAL4 BX transcript is mediated by MSK1/2 (mitogen- and stress-activated kinase 1/2) through the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and increases sLe(x) expression on high-molecular-mass glycoproteins in inflamed airway epithelium. We also show that the TNF-induced sLe(x) expression increases the adhesion of the Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells in a FliD-dependent manner. These results suggest that ERK and p38 MAPK, and the downstream kinase MSK1/2, should be considered as potential targets to hamper inflammation, bronchial mucin glycosylation changes and P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.

Knockdown of ZFX inhibits gastric cancer cell growth in vitro and in vivo via downregulating the ERK-MAPK pathway

Zinc finger protein X-linked (ZFX) is a zinc finger transcription factor encoded on the X chromosome. Here, we found that ZFX expression was significantly upregulated in gastric cancer (GC) cell lines and tissues. Knockdown of ZFX induced significant apoptosis and cell cycle arrest in SGC7901 and MGC803 cells. Moreover, we demonstrated for the first time that knockdown of ZFX inhibited gastric cancer cell growth in vitro and in vivo via downregulating the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway. Therefore, ZFX play a prominent role in GC tumorigenicity and may have potential applications in the diagnosis or treatment of GC.

Effect of inhibition of MEK pathway on 5-aza-deoxycytidine-suppressed pancreatic cancer cell proliferation

Pancreatic adenocarcinoma is a lethal disease because it is inoperable at the time of diagnosis. Therefore, the search for new therapeutic approaches is critical. The abnormal expression of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) pathway and alteration in epigenetic modification (DNA methylation and acetylation of histones) is a common feature in the majority of human pancreatic adenocarcinomas. Because DNA methyltransferase levels are regulated by the MEK pathway, we examined the effects of an MEK inhibitor, PD98059, on the action of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), the epigenetic agent in the pancreatic cell line CFPAC1. Our results showed that PD98059 significantly potentiated the capability of 5-aza-dC to induce a cessation of cell proliferation concomitant with cell cycle arrest. We also observed an increase in tumor suppressor gene expression associated with the efficacy of treatment with PD98059 and 5-aza-dC. Further studies explored the molecular mechanisms by which 5-aza-dC induced the expression of p21(WAF1). We found that 5-aza-dC induced acetylation of histone H3 on the p21(WAF1) gene promoter and demethylation status on the p21(WAF1) gene promoter region. These effects were strikingly enhanced by the concomitant blockade of the MEK pathway. Furthermore, knockdown of p21(WAF1) by small interfering RNA rescued human pancreatic cancer cells from 5-aza-dC-mediated growth inhibition. Taken together, our results show that the MEK inhibitor enhanced the effects of 5-aza-dC in human pancreatic cancer cells. These results suggest that the MEK signal pathway may be a potential target for pancreatic cancer therapy.

Biological Functions of Cytokeratin 18 in Cancer

The structural proteins cytokeratin 18 (CK18) and its coexpressed complementary partner CK8 are expressed in a variety of adult epithelial organs and may play a role in carcinogenesis. In this study, we focused on the biological functions of CK18, which is thought to modulate intracellular signaling and operates in conjunction with various related proteins. CK18 may affect carcinogenesis through several signaling pathways, including the phosphoinositide 3-kinase (PI3K)/Akt, Wnt, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathways. CK18 acts as an identical target of Akt in the PI3K/Akt pathway and of ERK1/2 in the ERK MAPK pathway, and regulation of CK18 by Wnt is involved in Akt activation. Finally, we discuss the importance of gaining a more complete understanding of the expression of CK18 during carcinogenesis, and suggest potential clinical applications of that understanding.

Lipopolysaccharide Enhances Decorin Expression through the Toll-like Receptor 4, Myeloid Differentiating Factor 88, Nuclear Factor-Kappa B, and Mitogen-activated Protein Kinase Pathways in Odontoblast Cells

IntroductionLipopolysaccharide (LPS) has been shown to regulate the function of odontoblasts. However, the molecular mechanisms of the effect of LPS on odontoblasts are poorly understood. Decorin (DCN), one of the major matrix proteoglycans, is known to affect the mineralization of teeth. In this study, we investigated whether LPS can regulate the expression of DCN in odontoblasts and determined the intracellular signaling pathways triggered by LPS. MethodsThe DCN messenger RNA and protein expression changes in mouse odontoblast-lineage cells (OLCs) were detected by real-time polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). Whether TLR4, myeloid differentiating factor 88 (MyD88), nuclear factor-kappa B (NF-κB), or mitogen-activated protein kinase (MAPK) pathways were involved in the LPS-induced DCN expression was determined by examined real-time PCR, ELISA, and luciferase activity assay. The activation of extracellular signal-regulated kinase (ERK), p38, and JNK in OLCs was measured by Western blot analysis. ResultsWe found that the mouse OLCs expressed DCN. DCN messenger RNA was rapidly induced by LPS in a time- and dose-dependent manner. Pretreatment with a MyD88 inhibitory peptide, a TLR4 antibody, or a specific inhibitor for NF-κB or I Kappa B alpha (IκBα) significantly inhibited LPS-induced DCN expression. Moreover, the LPS-mediated increase in κB-luciferase activity in OLCs was suppressed by the overexpression of dominant negative mutants of TLR4, MyD88, and IκBα but not by a dominant negative mutant of TLR2. In addition, LPS stimulation activated the ERK, p38, and JNK MAPK pathways. The pretreatment of OLCs with specific inhibitors of the ERK, p38, and JNK MAPK pathways markedly offset the LPS-induced up-regulation of DCN expression. ConclusionsOur results show that LPS stimulation can up-regulate the gene expression of DCN via the TLR4, MyD88, NF-κB, and MAPK pathways in odontoblast cells.

Immediate-early gene activation by the MAPK pathways: what do and don't we know?

The study of IE (immediate-early) gene activation mechanisms has provided numerous paradigms for how transcription is controlled in response to extracellular signalling. Many of the findings have been derived from investigating one of the IE genes, FOS, and the models extrapolated to regulatory mechanisms for other IE genes. However, whereas the overall principles of activation appear similar, recent evidence suggests that the underlying mechanistic details may differ depending on cell type, cellular stimulus and IE gene under investigation. In the present paper, we review recent advances in our understanding of IE gene transcription, chiefly focusing on FOS and its activation by ERK (extracellular-signal-regulated kinase) MAPK (mitogen-activated protein kinase) pathway signalling. We highlight important fundamental regulatory principles, but also illustrate the gaps in our current knowledge and the potential danger in making assumptions based on extrapolation from disparate studies.

IL-6/IL-6 receptor system and its role in physiological and pathological conditions

IL (interleukin)-6, which was originally identified as a B-cell differentiation factor, is a multifunctional cytokine that regulates the immune response, haemopoiesis, the acute phase response and inflammation. IL-6 is produced by various types of cell and influences various cell types, and has multiple biological activities through its unique receptor system. IL-6 exerts its biological activities through two molecules: IL-6R (IL-6 receptor) and gp130. When IL-6 binds to mIL-6R (membrane-bound form of IL-6R), homodimerization of gp130 is induced and a high-affinity functional receptor complex of IL-6, IL-6R and gp130 is formed. Interestingly, sIL-6R (soluble form of IL-6R) also binds with IL-6, and the IL-6-sIL-6R complex can then form a complex with gp130. The homodimerization of receptor complex activates JAKs (Janus kinases) that then phosphorylate tyrosine residues in the cytoplasmic domain of gp130. The gp130-mediated JAK activation by IL-6 triggers two main signalling pathways: the gp130 Tyr759-derived SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase-2)/ERK (extracellular-signal-regulated kinase) MAPK (mitogen-activated protein kinase) pathway and the gp130 YXXQ-mediated JAK/STAT (signal transducer and activator of transcription) pathway. Increased IL-6 levels are observed in several human inflammatory diseases, such as rheumatoid arthritis, Castleman's disease and systemic juvenile idiopathic arthritis. IL-6 is also critically involved in experimentally induced autoimmune diseases. All clinical findings and animal models suggest that IL-6 plays a number of critical roles in the pathogenesis of autoimmune diseases. In the present review, we first summarize the IL-6/IL-6R system and IL-6 signal transduction, and then go on to discuss the physiological and pathological roles of IL-6.

Erythroid differentiation regulator 1 (Erdr1) is a proapototic factor in human keratinocytes

Skin is constantly exposed to physical and chemical stressors. The exposure of keratinocytes to ultraviolet B (UVB) irradiation causes epidermal damage via induction of apoptosis. Erythroid differentiation regulator 1 (Erdr1) modulates growth and survival of cells under various stressful conditions, but the function of Erdr1 in human keratinocyte apoptosis has not been investigated so far. Here, we investigated the effect of Erdr1 on UVB-induced apoptosis in human keratinocytes and also examined the underlying regulatory mechanism. First, Erdr1 expression was detected in human primary keratinocytes and normal human skin tissues. Expression of Erdr1 was enhanced in human keratinocytes following UVB irradiation. Knock-down of Erdr1 led to resistance to UVB-induced apoptosis. Also, Erdr1 overexpression increased UVB-induced apoptosis and induced caspase-3 activation. Furthermore, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 significantly reduced Erdr1 expression following UVB irradiation. These results indicate that UVB induces Erdr1 via a MAPK-dependent mechanism. Taken together, these findings suggest that Erdr1 has a role as a proapoptotic factor in human keratinocytes and acts via ERK and p38 MAPK pathways. Therefore, Erdr1 may be a potential therapeutic target to reduce apoptosis in keratinocytes in conditions such as psoriasis and skin cancer.

Gadolinium inhibits prostate cancer PC3 cell migration and suppresses osteoclast differentiationin vitro

This study examined whether Gd (gadolinium) could suppress prostate cancer cell migration and prostate cancer cell-induced osteoclast differentiation. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] and colony forming assay showed that GdCl3 treatment inhibited both cell viability and colony forming ability in PC3 cells more significantly than that in DU145 cells. Annexin/PI (propidium iodide) staining showed an increase in apoptotic death of PC3 cells in the presence of GdCl3. Wound healing and adhesion assay indicated that GdCl3 suppressed PC3 cell migration. Western-blot analysis demonstrated that GdCl3 treatment inhibited phosphorylation of ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase). Pretreatment with PTx (pertussis toxin), a Gi protein inhibitor, conferred resistance to GdCl3-induced colony formation, ERK and p38 phosphorylation in PC3 cells. Moreover, GdCl3 inhibited PC3 cell-induced osteoclast differentiation. RT-PCR (reverse transcription-PCR) indicated that GdCl3 decreased the expression of RANKL (receptor activator of nuclear factor-κB ligand) in PC3 cells, whereas it increased the expression of OPG (osteoprotegerin) in PC3 and DU145 cells. In conclusion, the present study indicated that GdCl3 inhibited PC3 cell migration mediated by the inactivation of both ERK and p38 MAPK pathways via PTx-sensitive G proteins, and also suppressed PC3 cell-induced osteoclast differentiation via regulating the mRNA expression of OPG and RANKL.

Oncogenic role of EAPII in lung cancer development and its activation of the MAPK–ERK pathway

Cancer progression involves multiple complex and interdependent steps, including progressive proliferation, angiogenesis and metastases. The complexity of these processes requires a comprehensive elucidation of the integrated signaling networks for better understanding. EAPII interacts with multiple cancer-related proteins, but its biological significance in cancer development remains unknown. In this report we identified the elevated level of EAPII protein in non-small-cell lung carcinoma (NSCLC) patients and NSCLC cell lines in culture. The oncogenic role of EAPII in lung cancer development was demonstrated using NSCLC cells with genetic manipulations that influence EAPII expression: EAPII overexpression increases proliferation of NSCLC cells with an accelerated transition of cell cycle and facilitates xenograft tumor growth in vivo; EAPII knockdown results in apoptosis of NSCLC cells and reduces xenograft tumor formation. To further explore the mechanism of EAPII's oncogenic role in lung cancer development and to elucidate the potential signaling pathway(s) that EAPII may impact, we employed antibody array to investigate the alternation of the major signaling pathways in NSCLC cells with altered EAPII level. We found that EAPII overexpression significantly activated Raf1 and ERK1/2, but not c-Jun N-terminal kinase and p38 pathways. Consistently, the protein and mRNA levels of MYC and cyclin D1, which are targets of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK–ERK) pathway, are significantly increased by EAPII overexpression. Taken together, we demonstrated that EAPII is an oncogenic factor and the activation of MAPK–ERK signaling pathway by EAPII may contribute to lung cancer development.

4-hydroxy-2-nonenal upregulates and phosphorylates cytosolic phospholipase A2 in cultured Ra2 microglial cells via MAPK pathways

Several studies have suggested the involvement of neuroinflammation in the pathomechanism of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We recently demonstrated increased levels of protein-bound 4-hydroxy-2-nonenal (HNE) as a highly reactive lipid peroxidation product and cytosolic phospholipase A(2) (cPLA(2)) as a proinflammatory enzyme in glial cells as well as motor neurons in the spinal cord of sporadic ALS patients. However, a link between HNE and cPLA(2) in ALS remains to be determined. To address this issue, we investigated effects of HNE stimulation on the state of cPLA(2) expression in cultured microglial cell line (Ra2). Exposure of Ra2 cells to HNE significantly increased expression levels of cPLA(2) and its activated form phosphorylated at amino acid residue S(505) (p-cPLA(2)) on immunoblots. Pretreatment of Ra2 cells with the antioxidant N-acetylcysteine, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 prevented the HNE-induced increased expression of cPLA(2) and p-cPLA(2). Immunocytochemical analysis revealed that staining for p-cPLA(2) in Ra2 cells was localized in the cytoplasm and more intense in the HNE-stimulated group than in the vehicle group. The present results provide in vitro evidence that HNE upregulates and phosphorylates cPLA(2) in microglia via the ERK and p38 MAPK pathways.

Antagonistic roles of the ERK and p38 MAPK signalling pathways in globin expression, haem biosynthesis and iron uptake1

Late-stage erythroid cells synthesize large quantities of haemoglobin, a process requiring the co-ordinated regulation of globin and haem synthesis as well as iron uptake. In the present study, we investigated the role of the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in MEL (mouse erythroleukaemia) cell differentiation. We found that treatment of HMBA (hexamethylene bisacetamide)-induced MEL cells with the ERK pathway inhibitor UO126 results in an increase in intracellular haem and haemoglobin levels. The transcript levels of the genes coding for β(major)-globin, the haem biosynthesis enzyme 5-aminolevulinate synthase 2 and the mitochondrial iron transporter mitoferrin 1 are up-regulated. We also showed enhanced expression of globin and transferrin receptor 1 proteins upon UO126 treatment. With respect to iron uptake, we found that ERK inhibitor treatment led to an increase in both haem-bound and total iron. In contrast, treatment of MEL cells with the p38 MAPK pathway inhibitor SB202190 had the opposite effect, resulting in decreased globin expression, haem synthesis and iron uptake. Reporter assays showed that globin promoter and HS2 enhancer-mediated transcription was under the control of MAPKs, as inhibition of the ERK and p38 MAPK pathways led to increased and decreased gene activity respectively. Our present results suggest that the ERK1/2 and p38α/β MAPKs play antagonistic roles in HMBA-induced globin gene expression and erythroid differentiation. These results provide a novel link between MAPK signalling and the regulation of haem biosynthesis and iron uptake in erythroid cells.