Search

Both mitogen-activated protein kinase (MAPK) cascades and reactive oxygen species (ROS) are critical to signaling in eukaryotes. Cadmium induces MAPK activation and ROS production in plants. This study aims to identify specific MAPKs activated by CdCl2 in maize roots, and examine the relationship between MAPK activation and ROS production under CdCl2 treatment. Using in-gel kinase assays, immunoprecipitation, and immunoblot analysis, we identified 43 and 45 kDa ERK-like MAPKs in response to CdCl2. Further analysis revealed that ZmMPK3-1 and ZmMPK6-1 correspond to the 43 and 45 kDa MAPKs, respectively. In addition, CdCl2 induced ROS production prior to the activation of ZmMPK3-1 and ZmMPK6-1. Inhibition of ROS attenuated Cd-activation of ZmMPK3-1 and ZmMPK6-1, whereas inhibition of MAPK signaling did not disturb Cd-induced ROS production. Collectively, these results indicate that, in maize roots, cadmium stress activates ZmMPK3-1 and ZmMPK6-1 via ROS induction.

Mitogen-activated protein kinase (MAPK) cascades are involved in host defense against pathogens and are often activated by upstream plasma membrane leucine-rich repeat receptor-like kinases (LRR-RLKs). ERECTA (ER) is an LRR-RLK that regulates plant developmental processes through activating MAPK cascades. Tomato leaf curl Yunnan virus (TLCYnV) C4 protein interacts with BKI1, stabilizes it at the plasma membrane and impairs ER autophosphorylation through suppressing the dissociation of the BKI1/ER complex, and then inhibits the activation of downstream MAPK cascades, which ultimately creates a favorable environment for TLCYnV infection. This study provides a novel viral strategy to impair MAPK activation.

In the present study, we investigated the potential of opuntiol, isolated from Opuntia ficus-indica, against UVA radiation-mediated inflammation and skin photoaging in experimental animals. The skin-shaved experimental mouse was subjected to UVA exposure at the dosage of 10 J/cm2 per day for ten consecutive days (cumulative UVA dose: 100 J/cm2). Opuntiol (50 mg/kg b.wt.) was topically applied one hour before each UVA exposure. UVA (100 J/cm2) exposure induces epidermal hyperplasia and collagen disarrangement which leads to the photoaging-associated molecular changes in the mouse skin. Opuntiol pretreatment prevented UVA-linked clinical macroscopic skin lesions and histological changes in the mouse skin. Further, opuntiol prevents UVA-linked dermal collagen fiber loss in the mouse skin. Short-term UVA radiation (100 J/cm2) activates MAPKs through AP-1 and NF-κB p65 transcriptional pathways and subsequently induces the expression of inflammatory proteins and matrix-degrading proteinases in the mouse skin. Interestingly, opuntiol pretreatment inhibited UVA-induced activation of iNOS, VEGF, TNF-α, and COX-2 proteins and consequent activation of MMP-2, MMP-9, and MMP-12 in the mouse skin. Moreover, opuntiol was found to prevent collagen I and III breakdown in UVA radiation-exposed mouse skin. Thus, opuntiol protects mouse skin from UVA radiation-associated photoaging responses through inhibiting inflammatory responses, MAPK activation, and degradation of matrix collagen molecules.

Sarcomas are deadly malignant tumors of mesenchymal origin occurring at all ages. The expression and function of the membrane-type matrix metalloproteinase MMP14 is closely related to the mesenchymal cell phenotype, and it is highly expressed in most sarcomas. MMP14 regulates the activity of multiple extracellular and plasma membrane proteins, influencing cell–cell and cell–extracellular matrix (ECM) communication. This regulation mediates processes such as ECM degradation and remodeling, cell invasion, and cancer metastasis. Thus, a comprehensive understanding of the biology of MMP14 in sarcomas will shed light on the mechanisms controlling the key processes in these diseases. Here, we provide an overview of the function and regulation of MMP14 and we discuss their relationship with clinical and pre-clinical MMP14 data in both adult and childhood sarcomas. Published version

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival of 9% and effective treatment options remain elusive. Oncogenic mutations of KRAS occur in >95% of PDACs and are well-established as the bona fide driver event. However, inhibition of KRAS oncoprotein or its downstream signaling cascades remains unsuccessful in PDAC patients. Furthermore, parallel survival pathways including constitutive activation of the NF-κB pathway poses an additional therapeutic barrier. Previous work from our lab showed that Interleukin-1 Receptor associated kinase 4 (IRAK4) is a major driver of NF-kB cascade in PDAC. Here, through an unbiased reverse phase protein array screen and RNA sequencing, we discovered IRAK4 controls MAPK activity downstream of KRAS. Ablation of IRAK4 completely abolishes RAS-induced transformation in human and murine cell lines. Mechanistically, we implicate a KRAS-driven IL-1β signaling loop that activates IRAK4 and uncover MAP3K8 (or TPL2/COT) as the kinase through which IRAK4 activates MEK and ERK. Suppression of TPL2 abrogates KRAS-driven MEK-ERK activity and transformed growth of PDAC cell lines. In addition, TPL2 inhibition suppresses p105/p50 NF-kB activation, a valuable phenomenon that distinguishes TPL2 inhibition from MEK inhibition. We find TPL2 inhibition synergizes with chemotherapy to suppress growth of PDAC cell lines in vitro and patient-derived xenograft tumor model in vivo. Analyses of PDAC tissue microarray showed TPL2 expression to be marginally associated with poor prognosis. Additionally, we are the first to characterize gain-of-function point mutations in TPL2 which hyperactivate MAPK and NF-kB, in part by preventing TPL2 protein degradation. Together, our study broadens the understanding of the oncogenic RAS signaling network and reveals IRAK4 and TPL2 as novel practical therapeutic targets in RAS-driven cancers. Citation Format: Paarth B. Dodhiawala, Namrata Khurana, Daoxiang Zhang, Yi Cheng, Lin Li, Kuljeet Seehra, Hongmei Jiang, Patrick M. Grierson, Andrea Wang-Gillam, Marianna B. Ruzinova, Kian-Huat Lim. Essential role of IRAK4/TPL2 signaling axis in MAPK activation by oncogenic RAS and genotoxic stress [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3039.

Impaired wound healing and tissue regeneration have severe consequences on the patient’s quality of life. Micrograft therapies are emerging as promising and affordable alternatives to improve skin regeneration by enhancing the endogenous wound repair processes. However, the molecular mechanisms underpinning the beneficial effects of the micrograft treatments remain largely unknown. In this study, we identified the active protein-1 (AP-1) member Fos-related antigen-1 (Fra-1) to play a central role in the extracellular signal-regulated kinase- (ERK-) mediated enhanced cell migratory capacity of soluble micrograft-treated mouse adult fibroblasts and in the human keratinocyte cell model. Accordingly, we show that increased micrograft-dependent in vitro cell migration and matrix metalloprotease activity is abolished upon inhibition of AP-1. Furthermore, soluble micrograft treatment leads to increased expression and posttranslational phosphorylation of Fra-1 and c-Jun, resulting in the upregulation of wound healing-associated genes mainly involved in the regulation of cell migration. Collectively, our work provides insights into the molecular mechanisms behind the cell-free micrograft treatment, which might contribute to future advances in wound repair therapies. ispartof: Stem Cells International vol:2019 ispartof: location:United States status: Published online

The anti-neuroinflammatory effect of Astragaloside IV (ASI) has been reported, but its underlying mechanisms are unclear. This study is further to explore the underlying mechanism of ASI on anti-neuroinflammatory effect in LPS induced microglia cells. The result showed ASI significantly reduced the production of inflammatory mediators NO, TNF-α and IL-6 in BV2 and primary microglial cells. Western blot analysis showed ASI did not inhibit the MAPK activation, on the contrary, the results showed ASI can obviously induce the ERK activation. We also examined the NRF2 and HO-1 activation which were reported to exert anti-neuroinflammatory effect and the results presented it could induce the activation of HO-1 downstream NRF2 in BV2 microglial cells. Further study indicated the NRF2/HO-1 activation via ERK pathway activation. After NRF2 siRNA or HO-1 inhibitor treatment, the anti-neuroinflammatory effect of ASI was attenuated obviously compared with the normal group. Taken together, this study demonstrated that the activation of NRF2/HO-1 via ERK signaling pathway is a novel mechanism of ASI which exerted anti-neuroinflammatory activity in microglia cells, it could be an attractive candidate for the regulation of inflammatory responses in the brain.

Pathogen-associated molecular patterns (PAMPs) activate mitogen-activated protein kinases (MAPKs), essential components of plant defense signaling. Salicylic acid (SA) is also central to plant resistance responses, but its specific role in regulation of MAPK activation is not completely defined. We have investigated the role of SA in PAMP-triggered MAPKs pathways in Arabidopsis SA-related mutants, specifically in the flg22-triggered activation of MPK3 and MPK6. cim6, sid2, and npr1 mutants exhibited wild-type-like flg22-triggered MAPKs activation, suggesting that impairment of SA signaling has no effect on the flg22-triggered MAPKs activation. Pretreatment with low concentrations of SA enhanced flg22-induced MPK3 and MPK6 activation in all seedlings except npr1, indicating that NPR1 is involved in SA-mediated priming that enhanced flg22-induced MAPKs activation.

MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf Kinase Inhibitory Protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.

Suspension-cultured cells respond sensitively to a number of stimuli including pathogen-derived molecules. Therefore, they are used in a simple, single-cell model system in order to gain insights into immune signaling pathways in rice. Here we describe protocols for studying chitin-induced MAPK activation and ROS generation in rice suspension-cultured cells.

The NADPH oxidase (Nox) family of superoxide- and hydrogen peroxide—producing proteins has been recognized as important for signal transduction that regulates receptor-mediated functions, including cytoskeleton remodeling, cell proliferation, migration, differentiation, and cell death. Nox1 was the first of the Nox catalytic subunits to be cloned and shown to induce tumorigenic conversion of mouse fibroblasts. While Nox1 has been shown to be expressed in human colon and prostate cancers, however, limited studies have demonstrated the involvement of Nox1 in an early step of cell transformation. The aim of this review is to provide an overview on the role of Nox1 in cancer, as well as the contribution of our studies to demonstrate the involvement of Nox1 on neoplastic progression of human keratinocytes beyond the immortalization step. The generation of spindle phenotypes concomitant with anchorage-independent growth and invasiveness will be highlighted and discussed in relation to the possible role of Nox1 in epithelial-mesenchymal transition. Understanding these mechanisms may provide insight into Nox1 and redox signaling components as potential therapeutic targets to inhibit tumor progression.

Estrogen receptors (ERalpha and ERbeta) mediate opposite functions on cancer growth induced by 17beta-estradiol (E2). E2 binding to ERalpha induces a cancer promoting response, whereas E2 binding to ERbeta exerts a protective action against cancer growth. Moreover, E2 can diversely modulate the ERalpha and ERbeta levels intensifying or decreasing their actions in target tissues. Only molecular mechanisms at the root of E2 ability to down-regulate the ERalpha levels are known. Here, we report the first molecular mechanism underlying E2-induced ERbeta up-regulation in DLD-1 colon cancer cells. E2 induces a short term (2 and 4h after stimulation) translation of ERbeta mRNA followed by a late (24h after stimulation) enhanced transcription. Both processes required the E2-induced persistent and palmitoylation-dependent p38/MAPK activation. Overall, our data suggest a finely tuned control exerted by rapid signals on different cellular molecular events important for the protective effects of E2 against colon cancer growth.

We examined the impact of lymph flow obstruction in large post-nodal lymphatic vessels in sheep. A silk ligature was placed 2 cm downstream from the prescapular or popliteal lymph node and tightened to interrupt flow. At 6, 12 and 16 weeks after lymph flow blockage, a network of small interconnecting lymphatics (approximately 10-40 microm in diameter) could be observed in the vicinity of the ligature. These were identified using antibodies to the lymphatic endothelial markers LYVE-1 or VEGFR-3 or unequivocally, with the upstream intraluminal injection of the non-specific cell dye CFDA-SE. The observed lymphangiogenesis coincided with increased levels of Prox1, Tie2 (Y992) phosphorylation, MAPK activation, and decreased Akt activition. In the popliteal preparations, saline was infused into the prenodal ducts upstream of the regeneration site. The slopes of the inflow pressure versus flow relationships were 17.3+/-3.6, immediately after vessel obstruction, 36.2+/-9.6 at 6 weeks and 15.0+/-5.3 at 12-16 weeks. For comparison, the average slope in a completely intact popliteal system was 3.1+/-0.3 (from a previous publication). The resistance to flow remained high up to 12-16 weeks after flow obstruction suggesting that normal flow parameters had not been achieved over this time. The lymph node appeared to have some role in limiting the impact of post-nodal lymph obstruction, a function that appeared to be compromised by lymph stasis.

International audience; Abscisic acid (ABA) is a major phytohormone mediating important stress-related processes. We recently unveiled an ABA-activated MAPK signaling module constituted of MAP3K17/18-MKK3-MPK1/2/7/14. Unlike classical rapid MAPK activation, we showed that the activation of the new MAPK module is delayed and relies on the MAP3K protein synthesis. In this addendum, we discuss the role of this original and unexpected activation mechanism of MAPK cascades which suggests that MAPKs can regulate both early and long-term plant stress responses.

Signals transmitted by common components often elicit distinct (yet appropriate) outcomes. In yeast, two developmental options—mating and invasive growth—are both regulated by the same MAP kinase cascade. Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway. Kss1 has been thought to participate in mating only when Fus3 is absent. Instead, we show that Kss1 is rapidly phosphorylated and potently activated by mating pheromone in wild-type cells, and that this is required for normal pheromone-induced gene expression. Signal identity is apparently maintained because active Fus3 limits the extent of Kss1 activation, thereby preventing inappropriate signal crossover.

Mitogen-activated protein kinase (MAPK) activation provides cell type-specific signals important for cellular differentiation, proliferation, and survival. Cyclic AMP (cAMP) has divergent effects on MAPK activity depending on whether signaling is through Ras/Raf-1 or Rap1/B-raf. We found that central nervous system-derived neurons, but not astrocytes, express B-raf. In neurons, cAMP activated MAPK in a Rap1/B-raf-dependent manner, while in astrocytes, cAMP decreased MAPK activity. Inhibition of MAPK in neurons decreased neuronal growth factor-mediated survival, and activation of MAPK by cAMP analogues rescued neurons from death. Furthermore, constitutive expression of B-raf in astrocytoma cells increased MAPK activation, as seen in neurons, and enhanced proliferation. These data provide the first experimental evidence that B-raf is the molecular switch which dominantly permits differential cAMP-dependent regulation of MAPK in neurons versus astrocytes, with important implications for both survival and proliferation.

The nature of “toxic” tau in Alzheimer’s disease has been unclear. During pathogenesis, the importance of tau oligomerization versus tau phosphorylation is controversial and the investigation of both remains critical towards defining the “toxicity” of tau. The phosphorylation of tau on serines and/or threonines occurs early in the disease course and altering phosphorylation has been shown to disrupt neuropathogenesis. We have recently reported that in PC12-derived cells, tau had a role in signal transduction processes activated by NGF. By depleting tau, NGF-induced MAPK activation was attenuated and by restoring tau, MAPK activation was restored. Furthermore, the phosphorylation of tau on thr231 was required for tau to potentiate MAPK activation. Here we report the effects of additional disease-related tau phosphorylation sites and tau isoform on the ability of tau to potentiate MAPK activation. Our findings, which tested three other sites of phosphorylation, showed that phosphorylation at these other sites mainly lessened MAPK activation; none potentiated MAPK activation. In comparing 0N3R tau to the other five brain tau isoforms, most showed a trend towards less MAPK activation, with only 2N4R tau showing significantly less activation. Since MAPK activation has been reported in Alzheimer’s disease brain and is characteristic of cell proliferation mechanisms, tau phosphorylation that promotes MAPK activation could promote cell cycle activation mechanisms. In neurons, the activation of the cell cycle leads to cell death, suggesting that abnormally phosphorylated tau can be a toxic species. The relationship between tau oligomerization and its ability to potentiate MAPK activation needs to be determined.

We have previously demonstrated that, in COS-1 cells, inhibition of calmodulin increases Ras-GTP levels although it decreases Raf-1 activity and consequently MAPK. The present study analyzes the role of calmodulin in the regulation of Raf-1. First we show, using FRET microscopy, that inhibition of Raf-1 was not a consequence of a decreased interaction between H-Ras and Raf-1. Besides, the analysis of the phosphorylation state of Raf-1 showed that calmodulin, through downstream PI3K, is essential to ensure the Ser338-Raf-1 phosphorylation, critical for Raf-1 activation. We also show that the expression of a dominant negative mutant of PI3K impairs the calmodulin-mediated Raf-1 activation; in addition, both calmodulin and PI3K inhibitors decrease phospho-Ser338 and Raf-1 activity from upstream active H-Ras (H-RasG12V) and this effect is dependent on endocytosis. Importantly, in H-Ras depleted COS-1 cells, calmodulin does not modulate MAPK activation. Altogether, the results suggest that calmodulin regulation of MAPK in COS-1 cells relies upon H-Ras control of Raf-1 activity and involves PI3K.

The exposure to non-thermal microwave electromagnetic fields generated by mobile phones affects the expression of many proteins. This effect on transcription and protein stability can be mediated by the MAPK (mitogen-activated protein kinase) cascades, which serve as central signalling pathways and govern essentially all stimulated cellular processes. Indeed, long-term exposure of cells to mobile phone irradiation results in the activation of p38 as well as the ERK (extracellular-signal-regulated kinase) MAPKs. In the present study, we have studied the immediate effect of irradiation on the MAPK cascades, and found that ERKs, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities. Using signalling inhibitors, we delineated the mechanism that is involved in this activation. We found that the first step is mediated in the plasma membrane by NADH oxidase, which rapidly generates ROS (reactive oxygen species). These ROS then directly stimulate MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF [heparin-binding EGF (epidermal growth factor)]. This secreted factor activates the EGF receptor, which in turn further activates the ERK cascade. Thus this study demonstrates for the first time a detailed molecular mechanism by which electromagnetic irradiation from mobile phones induces the activation of the ERK cascade and thereby induces transcription and other cellular processes.

Mitogen-activated protein kinases (MAPKs), including p38 and c-Jun N-terminal kinase (JNK), have a key role in T cell receptor (TCR)-induced gene transcription but their precise mechanism of activation is not well understood. The findings of two recent papers provide new insight into the activation of p38 and JNK by the membrane-associated guanylate kinase (MAGUK) family members Dlgh1 and Carma1, respectively, and show how distinct MAGUK proteins control specific aspects of TCR-mediated MAPK activation.

In healthy hosts, acute infection with the opportunistic pathogen Toxoplasma gondii is controlled by innate production of IL-12, a key cytokine crucial for the development of protective immunity. Previous work has established that the mitogen-activated protein kinases (MAPK), particularly p38 and ERK1/2, are important regulators of T. gondii-induced IL-12 synthesis. Here we report that host cell Ca(2+) is required for activation of MAPK by T. gondii, as well as LPS and CpG, and for parasite-induced synthesis of IL-12. In addition, pharmacological mobilization of Ca(2+) stores in macrophages treated with parasites or LPS enhanced MAPK phosphorylation initiated by these stimuli. Investigation of the upstream mechanism by which Ca(2+) regulates MAPK activation revealed that T. gondii induced acute activation of conventional, Ca(2+)-dependent PKCalpha and PKCbeta, which are required for infection-induced MAPK activation and production of IL-12. Despite these findings, neither acute parasite infection nor LPS initiated a measurable Ca(2+) response in macrophages, suggesting that low levels of Ca(2+) are permissive for initiation of pro-inflammatory signaling. Together these data identify host cell Ca(2+) and PKC as crucial regulators of the innate immune response to microbial stimuli, including T. gondii.

The signal transduction mediating PAF-induced MAPK activation in CHO-cells stably expressing cloned PAF-receptor, has been extensively studied in our group (1). Pertussis toxin (PTX) blocked about 50% of the PAF-induced MAPK activation, without altering phosphatidylinositol (PI) hydrolysis and calcium responses. It was therefore speculated that the PAF-receptor mediates MAPK activation through one PTX-insensitive pathway that yields PI hydrolysis and possibly activation of conventional protein kinase C (cPKC), and another PTX-sensitive pathway that is likely to be independent of calcium and cPKC but mainly remains obscure.

Receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis is accompanied by intracellular Ca(2+) mobilization in a form of oscillations, which plays essential roles by activating sequentially Ca(2+)/calmodulin-dependent protein kinase, calcineurin and NFATc1, necessary in the osteoclast differentiation. However, it is not known whether Ca(2+) mobilization which is evoked in RANKL-independent way induces to differentiate into osteoclasts. In present study, we investigated Ca(2+) mobilization induced by aluminum fluoride (AlF4 (-)), a G-protein activator, with or without RANKL and the effects of AlF4 (-) on the osteoclastogenesis in primary cultured mouse bone marrow-derived macrophages (BMMs). We show here that AlF4 (-) induces intracellular Ca(2+) concentration ([Ca(2+)]i) oscillations, which is dependent on extracellular Ca(2+) influx. Notably, co-stimulation of AlF4 (-) with RANKL resulted in enhanced NFATc1 expression and formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells. Additionally, we confirmed that mitogen-activated protein kinase (MAPK) is also activated by AlF4 (-). Taken together, these results demonstrate that G-protein would be a novel modulator responsible for [Ca(2+)]i oscillations and MAPK activation which lead to enhancement of RANKL-mediated osteoclastogenesis.

Through live cell imaging and computational modeling, Neves et al. (2008) demonstrate that molecular interactions alone are insufficient to explain the patterns of MAPK activation: a model must include the cell morphology and spatial distribution of signaling molecules.