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The current study aimed to dissect the impacts and mechanisms of hydroxycamptothecin on breast cancer. Collect conditioned medium from MSCs cells to apply it into the co-culture system of breast cancer cells, which were pre-treated with hydroxycamptothecin. The cell counting kit was employed to measure the proliferation potential of cells, while the phosphorylation degrees of AKT/MAPKrelated proteins were examined via Western blotting. Then the cellular migration was test by transwell. Finally, the transcriptional and translational levels of IL-6 and RANTES in cells were detected by real-time PCR and enzyme-linked immunosorbent assay. HC could remarkably influence the interplay between MSC and breast malignant cells, reduce the MSC-activated migrative behavior of breast malignant cells and impede the capability of MSC to maintain the migration of cancer cells. RANTES and IL-6 exerted a synergistic induction in the migrative feature of breast cancer cells. HC could retard the migrating activities of breast cancer cells via diminishing the RANTES and IL-6 levels. Hydroxycamptothecin could impede the proliferative and migrative activities of MSC, of which the impediment was accompanied by an inhibitory impact on the secretory production of two growth factors IL-6 and RANTES from MSC, thereby enhancing the migration of breast malignant cells.

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.

The characteristic constituent and anti-inflammatory activity of 5- n-alkylresorcinols (ARs) from 21 wheat bran samples in China were investigated in this study. The amount of ARs ranged from 697 to 1732 μg/g in the tested samples, which were composed of five different homologues. Among these homologues, C19:0 and C21:0 were the most abundant, followed by C17:0, C23:0, and C25:0. Moreover, the mRNA expression of IL-1β, IL-6, and TNF-α in LPS-activated RAW264.7 macrophage cells were significantly inhibited by ARs supplementation. The molecular mechanisms behind its anti-inflammatory activity could result from the suppression of nuclear factor-κB (NF-κB) and JNK/MAPK activation. ARs treatment notably decreased NF-κB p65 nuclear translocation and inhibitor κB (IκBα) kinase and JNK phosphorylation. Additionally, ARs homologues C17:0 had been proven to be the main active constituent. The results from this study could be used to promote the comprehensive utilization of wheat and its byproducts in improving human health.

Ten new highly oxidized monomeric (1–8) and dimeric guaianolides (9 and 10), along with two known guaianolide derivatives (11 and 12), were isolated from the aerial parts of Chrysanthemum indicum using a bioassay-guided fractionation procedure. The new compounds were characterized by the basic analysis of the spectroscopic data obtained, and the absolute configurations were determined by both empirical approaches and ECD calculations. Inhibitory effects of 1–12 on nitric oxide production were investigated in lipopolysaccaride (LPS)-mediated RAW 264.7 cells, and most of them (1–8 and 11) displayed IC50 values in the range 1.4–9.7 μM. Moreover, a mechanistic study revealed that the potential anti-inflammatory activity of compound 1 appears to be mediated via suppression of an LPS-induced NF-κB pathway and down-regulation of MAPK activation.

Quinones are small secondary metabolites synthesized by a broad range of organisms. Perception of these aromatic molecules in plants involves membrane-bound LRR-RLKs to induce downstream cellular responses in plants such as calcium fluxes, specific gene expression and MAPK activation.

Optimal activation of TLR pathways is crucial for the initiation of inflammatory responses and eliminating invading micro-organisms. However, excessive of TLR activation may lead to autoimmune and inflammatory diseases. Thus, TLR pathways should be tightly controlled. In this study, we identify Tob2, a Tob/BTG family member, as a suppressor of TLR pathways. Tob2 deficiency enhances TLR-induced NF-κB and MAPK activation and promotes the expression of proinflammatory cytokines in primary peritoneal macrophages of C57BL/6 mice. Furthermore, Tob2-defective C57BL/6 mice may be more susceptible to endotoxemic shock in vivo. Mechanistically, Tob2 interacts with TRAF6 and MyD88 and thus inhibits signaling from the MyD88–TRAF6 complex in primary peritoneal macrophages and HEK293T cells. Therefore, our results uncover a regulatory mechanism of TLR pathways and provide a potential target for the intervention of diseases with excessive TLR 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.

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.

The purpose of the present study was to evaluate the anti-inflammatory effects of Portulaca oleracea L. (Purslane) extract on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and explore its potential mechanism. The RAW 264.7 cells were pretreated with Purslane extract for 4 h and subsequently induced by LPS for 12 h. It is of great concern that the bioactive compounds in Purslane extract and their metabolites in RAW 264.7 cell culture. What are the real bioactive compounds in the Purslaneextract that have the anti-inflammatory effects on RAW 264.7 cells, and when and how the bioactive compounds react? Based on this, 6 different groups were designed to be detected by LC/MS. (1): Purslane extract. (2): Blank medium without RAW 264.7 cells. (3): Medium pretreated by Purslane extract and induced by LPS without RAW 264.7 cells. (4): Blank medium in which RAW 264.7 cells were cultivated. (5): Medium induced by LPS in which RAW 264.7 cells were cultivated. (6): Medium pretreated by Purslane extract and induced by LPS in which RAW 264.7 cells were cultivated.

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.

Dynamic mechanical input is believed to play a critical role in the development of functional musculoskeletal tissues. To study this phenomenon, cyclic uniaxial mechanical stretch was applied to engineered ligaments using a custom-built bioreactor and the effects of different stretch frequency, amplitude, and duration were determined. Stretch acutely increased the phosphorylation of p38 (3.5±0.74-fold), S6K1 (3.9±0.19-fold), and ERK1/2 (2.45±0.32-fold). The phosphorylation of ERK1/2 was dependent on time, rather than on frequency or amplitude, within these constructs. ERK1/2 phosphorylation was similar following stretch at frequencies from 0.1 to 1 Hz and amplitudes from 2.5% to 15%, whereas phosphorylation reached maximal levels at 10 min of stretch and returned toward basal within 60 min of stretch. Following a single 10-min bout of cyclic stretch, the cells remained refractory to a second stretch for up to 6 h. Using the phosphorylation of ERK1/2 as a guide, the optimum stretch paradigm was hypothesized to be 10 min of stretch at 2.5% of resting length repeated every 6 h. Consistent with this hypothesis, 7 days of stretch using this optimized intermittent stretch program increased the collagen content of the grafts more than a continuous stretch program (CTL=3.1%±0.44%; CONT=4.8%±0.30%; and INT=5.9%±0.56%). These results suggest that short infrequent bouts of loading are optimal for improving engineered tendon and ligament physiology.

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.

INTRODUCTION: Osimertinib is a third-generation, mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) recently approved for the treatment of T790M-positive EGFR-mutant lung cancer. The mutant-selective nature of osimertinib improves its tolerability by limiting its inhibition of wild-type EGFR compared to first- and second-generation EGFR inhibitors, making it a good candidate for rationally-designed combination therapies. One such combination, osimertinib plus the MEK inhibitor selumetinib (AZD6244; ARRY-142886), is currently being studied as part of the phase 1b TATTON trial (NCT02143466). Previous work by our group demonstrated that selumetinib may delay or reverse resistance to osimertinib in some cases; however, we anticipate that resistance to this combination will ultimately develop in patients. Here, we investigate acquired resistance to osimertinib and selumetinib combination therapy in vitro and in vivo. DESIGN: In cells sensitive to osimertinib plus selumetinib (hereafter referred to as combination-sensitive cells), ERK phosphorylation was monitored via Western blot over the course of a 7-day treatment with 200 nM osimertinib plus 1 µM selumetinib. To detect RAS-GTP, an active RAS precipitation was performed in combination-sensitive and combination-resistant cell lines following treatment with osimertinib. In combination-resistant cells, pharmacologic inhibitors of various MAPK pathway components were used to assess whether they could restore potency. Western blot analysis was used to confirm on-target effects. Lastly, an analysis of differences in gene expression between four isogenic sets of combination-sensitive and combination-resistant cell lines is currently ongoing. RESULTS: In cell lines that are sensitive to growth inhibition by the combination of osimertinib plus selumetinib, we observed complete re-activation of the MAPK pathway after 5 days of continuous exposure to both inhibitors. Additionally, RAS activity was elevated in combination-resistant cell lines, and these cells remained sensitive to the ERK inhibitor SCH772984. Likewise, the addition of a pan-RAF inhibitor restored the growth inhibitory effects of osimertinib plus selumetinib in combination-resistant cells, suggesting that incomplete inhibition of MAPK is responsible for resistance in these cells. Furthermore, an alternative MEK inhibitor, trametinib, was efficacious in combination with osimertinib in cell lines that were resistant to osimertinib plus selumetinib. CONCLUSION: These data identify a potential mechanism of resistance to a combination therapy that is currently being tested in the clinic. Specifically, these data demonstrate that re-activation of the MAPK pathway is a mechanism of resistance to osimertinib plus selumetinib and that this pathway is still targetable in combination-resistant cells. Citation Format: David Westover, Catherine B. Meador, Eiki Ichihara, Hayden F. Byrd, Cath Eberlein, Yingjun Yan, Darren A. Cross, Christine M. Lovly. Sustained MAPK activation as a mechanism of resistance to osimertinib plus selumetinib in models of EGFR-mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3160. doi:10.1158/1538-7445.AM2017-3160

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.

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.

Alcohol consumption along with smoking is a well-known health hazard to human. How alcohol consumption potentiates the smoking mediated hazard is not clear. Previously we reported that DNA damage caused by tobacco smoke carcinogen (+/-) anti-benzo[a]pyrene-7, 8-diol-9,10-epoxide [BPDE] is responded by cells through induction of cell growth inhibition in different cell lines. Here we observed that ethanol (EtOH) treatment at physiologically relevant concentration (60mM) increases the DNA synthesis in DNA damaging carcinogen (BPDE) treated cells. BPDE treatment of cells elicits G1-S cell cycle arrest, but EtOH does not have any significant modulating effect of G1-S arrest, indicating increased DNA synthesis by EtOH is due to modulation of some other signaling event(s). We observed that EtOH co-treatment/ pretreatment potentiates BPDE induced phosphorylation and activation of extracellular regulated kinase 1/2 (ERK1/2). Interestingly, treatment of cells with MEK1 inhibitor (PD09059) significantly reduced EtOH's ability to increase DNA synthesis in BPDE treated cells. All together, these findings suggest that the ability of EtOH to potentiate BPDE-induced ERK activation may potentiates PAH-induced tumorigenesis. [Supported by NIH grant # R03 ES021779]. Citation Format: Manoj K. Pandey, Jagat J. Mukherjee, Dhimant H. Desai, Shantu G. Amin, Subodh Kumar. Ethanol potentiates tobacco smoke carcinogens-induced MAPK activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2734. doi:10.1158/1538-7445.AM2015-2734

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.

Erratum Erratum to “5-Hydroxytryptamine induces cyclooxygenase-2 in rat vascular smooth muscle cells: Mechanisms involving Src, PKC and MAPK activation” [Eur. J. Pharmacol. 656 (2011) 19-26] Takuji Machida, Misa Ohta, Akina Onoguchi, Kenji Iizuka, Mio Sakai, Masaru Minami, Masahiko Hirafuji⁎ Department of Pharmacological Sciences, School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061–0293, Japan

ISEE 22nd Annual Conference, Seoul, Korea, 28 August-1 September 2010: Heavy Metals and Metalloids