The crosstalk between Wnt/β-catenin signaling pathway with DNA damage response and oxidative stress: Implications in cancer therapy

DNA repair is essential for maintaining genomic integrity in cells. The dependence of cancer cell survival on proper DNA repair provides an opportunity to treat defective tumors by DNA damaging agents. Not only Wnt signaling has important functions in controlling gene expression, as well as cell polarity, adhesion and behavior, it also highly interacts with DNA damage response (DDR) in different levels. Furthermore, oxidative stress, which is responsible for majority of DNA lesions, affects Wnt signaling in different ways. A better understanding of the cross-talk between these pathways and events could provide strategies for treatment of cancer cells with deficient DNA repair capacity. As such, we will give a brief overview of the importance of the DNA repair machinery, signaling mechanisms of Wnt/β-catenin pathway, and DDR. We will further review the interactions between Wnt signaling and DDR, and the impact of oxidative stress on Wnt signaling. Finally, Wnt signaling is discussed as a potential treatment strategy for cancer.     

Pathological roles of MAPK signaling pathways in human diseases

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH₂-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.     

Autophagy and genomic integrity

DNA lesions, constantly produced by endogenous and exogenous sources, activate the DNA damage response (DDR), which involves detection, signaling and repair of the damage. Autophagy, a lysosome-dependent degradation pathway that is activated by stressful situations such as starvation and oxidative stress, regulates cell fate after DNA damage and also has a pivotal role in the maintenance of nuclear and mitochondrial genomic integrity. Here, we review important evidence regarding the role played by autophagy in preventing genomic instability and tumorigenesis, as well as in micronuclei degradation. Several pathways governing autophagy activation after DNA injury and the influence of autophagy upon the processing of genomic lesions are also discussed herein. In this line, the mechanisms by which several proteins participate in both DDR and autophagy, and the importance of this crosstalk in cancer and neurodegeneration will be presented in an integrated fashion. At last, we present a hypothetical model of the role played by autophagy in dictating cell fate after genotoxic stress.     

Docosahexaenoic acid prevents apoptosis of retina photoreceptors by activating the ERK/MAPK pathway

Identifying the trophic factors for retina photoreceptors and the intracellular pathways activated to promote cell survival is crucial for treating retina neurodegenerative diseases. Docosahexaenoic acid (DHA), the major retinal polyunsaturated fatty acid, prevents photoreceptor apoptosis during early development in vitro , and upon oxidative stress. However, the signaling mechanisms activated by DHA are still unclear. We investigated whether the extracellular signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) or the phosphatidylinositol-3-kinase (PI3K) pathway participated in DHA protection. 1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophynyltio) butadiene (U0126), a specific MEK inhibitor, completely blocked the DHA anti-apoptotic effect. DHA rapidly increased ERK phosphorylation in photoreceptors, whereas U0126 blocked this increase. U0126 hindered DHA prevention of mitochondrial depolarization, and blocked the DHA-induced increase in opsin expression. On the contrary, PI3K inhibitors did not diminish the DHA protective effect. DHA promoted the early expression of Bcl-2, decreased Bax expression and reduced caspase-3 activation in photoreceptors. These results suggest that DHA exclusively activates the ERK/MAPK pathway to promote photoreceptor survival during early development in vitro and upon oxidative stress. This leads to the regulation of Bcl-2 and Bax expression, thus preserving mitochondrial membrane potential and inhibiting caspase activation. Hence, DHA, a lipid trophic factor, promotes photoreceptor survival and differentiation by activating the same signaling pathways triggered by peptidic trophic factors.     

Hyperoxia induces Egr-1 expression through activation of extracellular signal-regulated kinase 1/2 pathway

Early growth response gene (Egr-1) is a stress response gene activated by various forms of stress and growth factor signaling. We report that supraphysiologic concentrations of O(2) (hyperoxia) induced Egr-1 mRNA and protein expression in cultured alveolar epithelial cells, as well as in mouse lung in vivo. The contribution of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), p38 MAPK and PI3-kinase pathways to the activation of Egr-1 in response to hyperoxia was examined. Exposure to hyperoxia resulted in a rapid phosphorylation of ERK 1/2 kinases in mouse alveolar epithelial cells LA4. MEK inhibitor PD98059, but not inhibitors of p38 MAPK or PI3-kinase pathway, prevented Egr-1 induction by hyperoxia. The signaling cascade preceding Egr-1 activation was traced to epidermal growth factor receptor (EGFR) signaling. Hyperoxia is used as supplemental therapy in some diseases and typically results in elevated levels of reactive oxygen intermediates (ROI) in many lung cell types, the organ that receives highest O(2) exposure. Our results support a pathway for the hyperoxia response that involves EGF receptor, MEK/ERK pathway, and other unknown signaling components leading to Egr-1 induction. This forms a foundation for analysis of detailed mechanisms underlying Egr-1 activation during hyperoxia and understanding its consequences for regulating cell response to oxygen toxicity.     

Activation of extracellular signal-regulated protein kinase 5 downregulates FasL upon osmotic stress

Extracellular signal-regulated protein kinase (ERK) 5 is a mitogen-activated protein kinase (MAPK) that is activated by dual phosphorylation via a unique MAPK/ERK kinase 5, MEK5. The physiological importance of this signaling cascade is underscored by the early embryonic death caused by the targeted deletion of the erk5 or the mek5 genes in mice. Here, we have found that ERK5 is required for mediating the survival of fibroblasts under basal conditions and in response to sorbitol treatment. Increased Fas ligand (FasL) expression acts as a positive feedback loop to enhance apoptosis of ERK5- or MEK5-deficient cells under conditions of osmotic stress. Compared to wild-type cells, erk5-/- and mek5-/- fibroblasts treated with sorbitol display a reduced protein kinase B (PKB) activity associated with increased Forkhead box O3a (Foxo3a) activity. Based on these results, we conclude that the ERK5 signaling pathway promotes cell survival by downregulating FasL expression via a mechanism that implicates PKB-dependent inhibition of Foxo3a downstream of phosphoinositide 3 kinase.     

Bcl‐2‐associated athanogene 5 overexpression attenuates catecholamine‐induced vascular endothelial cell apoptosis

Bcl‐2 associated athanogene 5 (Bag5) is a novel endoplasmic reticulum (ER) regulator. However, its role in catecholamine‐induced endothelial cells damage has not been fully understood. In our study, catecholamine was used to mimic hypertension‐related endothelial cell damage. Then, western blots, enzyme‐linked immunosorbent assay, immunofluorescence, quantitative polymerase chain reaction and pathway analysis were conducted to analyze the role of Bag5 in endothelial cell damage in response to catecholamine. Our results indicated that the endothelial cell viability was impaired by catecholamine. Interestingly, Bag5 overexpression significantly reversed endothelial cell viability. Mechanistically, Bag5 overexpression inhibited ER stress, attenuated oxidative stress and repressed inflammation in catecholamine‐treated endothelial cells. These beneficial effects finally contributed to endothelial cell survival under catecholamine treatment. Pathway analysis demonstrated that Bag5 was under the control of the mitogen‐activated protein kinase (MAPK)–extracellular‐signal‐regulated kinase (ERK) signaling pathway. Reactivation of the MAPK–ERK pathway could upregulate Bag5 expression and thus promote endothelial cell survival through inhibiting oxidative stress, ER stress, and inflammation. Altogether, our results illustrate that Bag5 overexpression sustains endothelial cell survival in response to catecholamine treatment. This finding identifies Bag5 downregulation and the inactivated MAPK–ERK pathway as potential mechanisms underlying catecholamine‐induced endothelial cell damage.     

E3 ligases and deubiquitinating enzymes regulating the MAPK signaling pathway in cancers

The mitogen-activated protein kinase (MAPK) signaling pathway is the primary regulatory module of various cellular processes such as cell proliferation, differentiation, and stress responses. This pathway converts external stimuli to cellular responses via three major kinases: mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase kinase kinase (MAPKKK). Ubiquitination is a post-translational modification of proteins with ubiquitin, which results in the formation of mono- or poly-ubiquitin chains of substrate proteins. Conversely, removal of the ubiquitin by deubiquitinating enzymes (DUBs) is known as deubiquitination. This review summarizes mechanisms of the MAPK signaling pathways (ERK1/2, ERK5, p38, and JNK1/2/3 signaling pathway) in cancers, and of E3 ligases and DUBs that target the MAPK signaling components such as Raf, MEK1/2, ERK1/2, MEKK2/3, MEKK1-4, TAK1, DLK1, MLK1-4, ASK1/2, and MKK3-7.     

Control of MAP kinase signaling to the nucleus

MAP kinase (MAPK) signaling is among central signaling pathways that regulate cell proliferation, cell differentiation and apoptosis. As MAPK should transmit extracellular signals to proper regions or compartments in cells, controlling subcellular localization of MAPK is important for regulating fidelity and specificity of MAPK signaling. The ERK1/2-type of MAPK is the best characterized member of the MAPK family. In response to extracellular stimulus, ERK1/2 translocates from the cytoplasm to the nucleus by passing through the nuclear pore by several independent mechanisms. Sef (similar expression to fgf genes), a transmembrane protein, has been shown to be a regulator of subcellular distribution of ERK1/2. Sef binds to activated MEK1/2, the specific activator of ERK1/2, and tethers the activated MEK1/2/activated ERK1/2 complex to the Golgi apparatus and the plasma membrane. Thus, Sef blocks ERK1/2 signaling to the nucleus and allows signaling to the cytoplasm. Here we review recent findings on spatial regulation of MAPK, especially on nucleocytoplasmic trafficking of ERK1/2.     

D1 dopamine receptor mediates dopamine-induced cytotoxicity via the ERK signal cascade

Postsynaptic striatal neurodegeneration occurs through unknown mechanisms, but it is linked to high extracellular levels of synaptic dopamine. Dopamine-mediated cytotoxicity of striatal neurons occurs through two distinct pathways: autoxidation and the D1 dopamine receptor-linked signaling pathway. Here we investigated the mitogen-activated protein kinase (MAPK) signaling pathways activated upon the acute stimulation of D1 dopamine receptors. In SK-N-MC neuroblastoma cells, endogenously expressing D1 dopamine receptors, dopamine caused activation of phosphorylated (p-)ERK1/2 and of the stress-signaling kinases, p-JNK and p-p38 MAPK, in a time- and dose-dependent manner. Selective stimulation of D1 receptors with the agonist SKF R-38393 caused p-ERK1/2, but not p-JNK or p-p38 MAPK activation, in a manner sensitive to the receptor-selective antagonist SCH 23390, protein kinase A inhibition (KT5720), and MEK1/2 inhibition (U0126 or PD98059). Activation of ERK by D1 dopamine receptors resulted in oxidative stress and cytotoxicity. In cells transfected with a catalytically defective mutant of MEK1, the upstream ERK-specific kinase, both dopamine- and SKF R-38393-mediated cytotoxicity was markedly attenuated, confirming the participation of the ERK signaling pathway. Cell fractionation studies showed that only a small amount of p-ERK1/2 was translocated to the nucleus, with the majority retained in the cytoplasm. From coimmunoprecipitation studies, p-ERK was found to form stable heterotrimeric complexes with the D1 dopamine receptor and beta-arrestin2. In cells transfected with the dominant negative mutant of beta-arrestin2, the formation of such complexes was substantially inhibited. These data provide novel mechanistic insights into the role of ERK in the cytotoxicity mediated upon activation of the D1 dopamine receptor.     

Time Courses of Changes in Phospho- and Total- MAP Kinases in the Cochlea after Intense Noise Exposure

Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute sensorineural hearing loss.     

Involvement of 3-phosphoinositide-dependent protein kinase-1 in the MEK/MAPK signal transduction pathway

The phosphatidylinositide-3-OH kinase/3-phospho-inositide-dependent protein kinase-1 (PDK1)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Despite their importance, however, the cross-talk between these two pathways has not been fully understood. Here we report that PDK1 promotes MAPK activation in a MEK-dependent manner. In vitro kinase assay revealed that the direct targets of PDK1 in the MAPK pathway were the upstream MAPK kinases MEK1 and MEK2. The identified PDK1 phosphorylation sites in MEK1 and MEK2 are Ser222 and Ser226, respectively, and are known to be essential for full activation. To date, these sites are thought to be phosphorylated by Raf kinases. However, PDK1 gene silencing using small interference RNA demonstrates that PDK1 is associated with maintaining the steady-state phosphorylated MEK level and cell growth. The small interference RNA-mediated down-regulation of PDK1 attenuated maximum MEK and MAPK activities but could not prolong MAPK signaling duration. Stable and transient expression of constitutively active MEK1 overcame these effects. Our results suggest a novel cross-talk between the phosphatidylinositide-3-OH kinase/PDK1/Akt pathway and the Raf/MEK/MAPK pathway.     

Divergent Requirements for the MAPKERK Signal Transduction Pathway during Initial Virus Infection of Quiescent Primary B Cells and Disruption of Epstein-Barr Virus Latency by Phorbol Esters

Quiescent primary B lymphocytes and Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines express components of the extracellular response kinase arm of the mitogen-activated protein kinase (MAPK(ERK)) signal transduction pathway and transmit signals through the pathway when exposed to appropriate stimuli. Although the MAPK(ERK) pathway is activated following infection with EBV, MAPK/ERK kinase (MEK1) activity is not required to drive the proliferation of infected cells. However, MEK1 contributes to EBV latency control.     

Endosomal targeting of MEK2 requires RAF, MEK kinase activity and clathrin-dependent endocytosis

To study spatiotemporal regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2) signaling cascade in living cells, a HeLa cell line in which MAPK kinase of ERK kinase (MEK) 2 (MAPK kinase) was knocked down by RNA interference and replaced with the green fluorescent protein (GFP)-tagged MEK2 was generated. In these cells, MEK2-GFP was stably expressed at a level similar to that of the endogenous MEK2 in the parental cells. Upon activation of the EGF receptor (EGFR), a pool of MEK2-GFP was found initially translocated to the plasma membrane and then accumulated in a subset of early and late endosomes. However, activated MEK was detected only at the plasma membrane and not in endosomes. Surprisingly, MEK2-GFP endosomes did not contain active EGFR, suggesting that endosomal MEK2-GFP was separated from the upstream signaling complexes. Knockdown of clathrin by small interfering RNA (siRNA) abolished MEK2 recruitment to endosomes but resulted in increased activation of ERK without affecting the activity of MEK2-GFP. The accumulation of MEK2-GFP in endosomes was also blocked by siRNA depletion of RAF kinases and by the MEK1/2 inhibitor, UO126. We propose that the recruitment of MEK2 to endosomes can be a part of the negative feedback regulation of the EGFR-MAPK signaling pathway by endocytosis.     

Role of the amino-terminal domains of MEKKs in the activation of NF kappa B and MAPK pathways and in the regulation of cell proliferation and apoptosis.

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) kinases (MEKKs) are serine/threonine kinases that are upstream regulators of MAPKs. Here, the role of the amino-terminal (N-terminal) domain of MEKK1-4 on the regulation of different intracellular signaling pathways, apoptosis, and cell proliferation has been assessed by comparing the responses induced by the full-length (FL) MEKKs to those induced by the kinase domains only. For each MEKK, the pattern of activation of NF kappa B, the ERK MAPK pathway, and the c-Jun N-terminal kinase (JNK) MAPK pathway markedly differed between the kinase domain and the FL form. Similarly, cell proliferation and apoptosis were differently regulated by the FL MEKK and the corresponding kinase domain. Our data show that the N-terminal domain of the MEKKs determines the specificity and the strength of activation of various intracellular signaling pathways and cellular responses.     

Oligonol, an oligomerized lychee fruit-derived polyphenol, activates the Ras/Raf-1/MEK1/2 cascade independent of the IL-6 signaling pathway in rat primary adipocytes

Oligonol is a lychee fruit-derived low-molecular form of polyphenol. In this study, the effect of Oligonol on the mitogen activated-protein kinase (MAPK) signaling pathway in primary adipocytes was investigated to examine the mechanism underlying the enhanced levels of phosphorylated extracellular-signaling regulatory kinase1/2 (ERK1/2) that accompany an in vitro increase in lipolysis. Oligonol significantly elevated the levels of activated Ras and the phosphorylation of Raf-1 and MAPK/ERK kinase1/2 (MEK1/2) with no increase in pan-Raf-1 and -MEK1/2 proteins. The increase in phosphorylation of Raf-1 and MEK1/2 with Oligonol was inhibited completely by pretreatment with GW5074, a selective Raf-1 inhibitor, or PD98059, a selective MEK1/2 inhibitor. IL-6 also activated the MAPK signaling pathway in adipocytes through the association with its receptor. IL-6-induced phosphorylation of Raf-1 and MEK1/2 was significantly inhibited by pretreatment with the IL-6 receptor antibody. Under such a condition, however, the levels of phosphorylated Raf-1 and MEK1/2 with Oligonol still remained significantly higher, and there was a significant decrease in secretion of IL-6 from adipocytes, compared with untreated control cells. These results suggest that Oligonol activates the Ras/Raf-1/MEK1/2 signaling pathway, independent of the IL-6 signaling pathway, leading to activation of ERK1/2 proteins in primary adipocytes.     

The roles of p27Kip1 and DNA damage signalling in the chemotherapy‐induced delayed cell cycle checkpoint

DNA lesions trigger the DNA damage response (DDR) machinery, which protects genomic integrity and sustains cellular survival. Increasing data underline the significance of the integrity of the DDR pathway in chemotherapy response. According to a recent work, persistent exposure of A549 lung carcinoma cells to doxorubicin induces an initial DDR‐dependent checkpoint response, followed by a later DDR‐independent, but p27^Kip1‐dependent one. Prompted by the above report and to better understand the involvement of the DDR signaling after chemotherapeutic stress, we examined the potential role of the canonical DDR pathway in A549 cells treated with doxorubicin. Exposure of A549 cells, prior to doxorubicin treatment, to ATM, ATR and DNA‐PKcs inhibitors either alone or in various combinations, revealed that the earlier documented two‐step response was DDR‐dependent in both steps. Notably, inhibition of both ATM and ATR or selective inhibition of ATM or DNA‐PKcs resulted in cell‐cycle re‐entry despite the increased levels of p27^Kip1 at all time points analyzed. We further investigated the regulation of p27^Kip1 protein levels in the particular setting. Our results showed that the protein status of p27^Kip1 is mainly determined by p38‐MAPK, whereas the role of SKP2 is less significant in the doxoroubicin‐treated A549 cells. Cumulatively, we provide evidence that the DNA damage signaling is responsible for the prolonged cell cycle arrest observed after persistent chemotherapy‐induced genotoxic stress. In conclusion, precise identification of the molecular mechanisms that are activated during the chemotherapeutic cycles could potentially increase the sensitization to the therapy applied.     

The Hog1 MAP kinase pathway and the Mec1 DNA damage checkpoint pathway independently control the cellular responses to hydrogen peroxide

The DNA damage checkpoint pathway and the MAP kinase pathway respond to various forms of environmental as well as endogenous stresses through signal transduction mechanisms involving protein kinases. Both pathways are intertwined in mammalian cells, but potential crosstalk between these two pathways in budding yeast has not been examined yet. We show that the Rad53 checkpoint kinase and the Hog1 MAP kinase of Saccharomyces cerevisiae become phosphorylated upon exposure to hydrogen peroxide, indicative of activation of the DNA damage checkpoint and MAP kinase pathways in response to oxidative stress. Rad53 kinase is equally activated in wild type and in hog1-Delta cells. Likewise, the activation of Hog1 MAP kinase is not dependent on Mec1 kinase, the central checkpoint kinase in budding yeast. Mutants in either pathway are sensitive to hydrogen peroxide and the double mutants exhibit a near perfectly additive phenotype. These data demonstrate that the DNA damage checkpoint pathway and the MAP kinase pathway respond to oxidative stress independently of each other and suggest that these two stress signaling pathways are activated by different types of insults induced by hydrogen peroxide.