Inhibition Of MAPK P38 Research Articles

Chemical Profiling and Bioactivity of Body Wall Lipids from Strongylocentrotus droebachiensis.

The lipids from gonads and polyhydroxynaphthoquinone pigments from body walls of sea urchins are intensively studied. However, little is known about the body wall (BW) lipids. Ethanol extract (55 °C) contained about equal amounts of saturated (SaFA) and monounsaturated fatty acids (MUFA) representing 60% of total fatty acids, with myristic, palmitic and eicosenoic acids as major SaFAs and MUFAs, respectively. Non-methylene-interrupted dienes (13%) were composed of eicosadienoic and docosadienoic acids. Long-chain polyunsaturated fatty acids (LC-PUFA) included two main components, n6 arachidonic and n3 eicosapentaenoic acids, even with equal concentrations (15 μg/mg) and a balanced n6/n3 PUFA ratio (0.86). The UPLC-ELSD analysis showed that a great majority of the lipids (80%) in the ethanolic extract were phosphatidylcholine (60 μg/mg) and phosphatidylethanolamine (40 μg/mg), while the proportion of neutral lipids remained lower than 20%. In addition, alkoxyglycerol derivatives—chimyl, selachyl, and batyl alcohols—were quantified. We have assumed that the mechanism of action of body wall lipids in the present study is via the inhibition of MAPK p38, COX-1, and COX-2. Our findings open the prospective to utilize this lipid fraction as a source for the development of drugs with anti-inflammatory activity.

Modulation by Central MAPKs/PI3K/sGc of the TNF-α/iNOS-dependent Hypotension and Compromised Cardiac Autonomic Control in Endotoxic Rats.

Reduced blood pressure (BP) and cardiac autonomic activity are early manifestations of endotoxemia. We investigated whether these effects are modulated by central mitogen-activated protein kinases (MAPKs) and related phosphoinositide-3-kinase (PI3K)/soluble guanylate cyclase (sGC) signaling in conscious rats. The effect of pharmacologic inhibition of these molecular substrates on BP, heart rate (HR), and heart rate variability (HRV) responses evoked by intravascular lipopolysaccharide (LPS) (10 mg/kg) were assessed. LPS (1) lowered BP (2) increased HR, (3) reduced time [SD of beat-to-beat intervals (SDNN), and root mean square of successive differences in R-R intervals (rMSSD)], and frequency domain indices of HRV (total power and spectral bands of low and high-frequency), and (4) elevated serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels. The inhibition of TNF-α (pentoxifylline) or inducible nitric oxide synthase (iNOS, aminoguanidine) abolished hemodynamic, HRV, and inflammatory actions of LPS. Intracisternal (i.c.) injection of ODQ (sGC inhibitor), wortmannin (PI3K inhibitor), and SP600125 (MAPKJNK inhibitor) mitigated the hypotensive and tachycardic actions of LPS but failed to affect associated decreases in HRV. MAPKp38 inhibition by i.c. SB203580 produced exactly opposite effects. None of the LPS effects was altered after i.c. PD98059 (MAPKERK1/2 inhibitor). Overall, central MAPKs/PI3K/sGC pathways variably contribute to the TNF-α/iNOS-dependent reductions in BP and HRV seen during endotoxic shock.

Regulation of S100B Expression in Long-Term Potentiation

In this study, contributions of intracellular regulatory cascades in the induction of S100B expression in rat hippocampal CA1 area during long term posttetanic potentiation (LTP) were estimated. The activation of transcription factor p53 (positive regulator of S100B transcription) by nutlin-3 increased the basal content of S100B mRNA up to 151% of the control level, which was significantly lower than its content in tetanized slices (280%). Therefore, p53 seems to be not unique transcription factor upregulating S100B expression during LTP. The inhibitor of Ca2+/calmodulin-dependent kinases (CaMKs) KN-93 fully blocked the increase of S100B mRNA after tetanization, while KN-92 (inactive analogue of KN-93) was ineffective. The inhibitor of CaMKII and receptor tyrosine kinases K-252a essentially suppressed S100B expression during LTP, the inhibition of MAPK p38 or RSK2 moderately decreased, and the inhibition of MEK1 did not influence S100B mRNA content. Thus, CaMKs play a key role in the induction of S100B expression during LTP.

Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population

The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD). Rhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium. MEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells. Our results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance. Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma.

The estrogen-dependent baroreflex dysfunction caused by nicotine in female rats is mediated via NOS/HO inhibition: Role of sGC/PI3K/MAPKERK

We have previously reported that estrogen (E2) exacerbates the depressant effect of chronic nicotine on arterial baroreceptor activity in female rats. Here, we tested the hypothesis that this nicotine effect is modulated by nitric oxide synthase (NOS) and/or heme oxygenase (HO) and their downstream soluble guanylate cyclase (sGC)/phosphatidylinositol 3-kinase (PI3K)/mitogen-activated protein kinases (MAPKs) signaling. We investigated the effects of (i) inhibition or facilitation of NOS or HO on the interaction of nicotine (2mg/kg/dayi.p., 2weeks) with reflex bradycardic responses to phenylephrine in ovariectomized (OVX) rats treated with E2 or vehicle, and (ii) central pharmacologic inhibition of sGC, PI3K, or MAPKs on the interaction. The data showed that the attenuation by nicotine of reflex bradycardia in OVXE2 rats was abolished after treatment with hemin (HO inducer) or l-arginine (NOS substrate). The hemin or l-arginine effect disappeared after inhibition of NOS (Nω-Nitro-l-arginine methyl ester hydrochloride, L-NAME) and HO (zinc protoporphyrin IX, ZnPP), respectively, denoting the interaction between the two enzymatic pathways. E2-receptor blockade (ICI 182,780) reduced baroreflexes in OVXE2 rats but had no effect on baroreflex improvement induced by hemin or l-arginine. Moreover, baroreflex enhancement by hemin was eliminated following intracisternal (i.c.) administration of wortmannin, ODQ, or PD98059 (inhibitors of PI3K, sGC, and extracellular signal-regulated kinases, MAPKERK, respectively). In contrast, the hemin effect was preserved after inhibition of MAPKp38 (SB203580) or MAPKJNK (SP600125). Overall, NOS/HO interruption underlies baroreflex dysfunction caused by nicotine in female rats and the facilitation of NOS/HO-coupled sGC/PI3K/MAPKERK signaling might rectify the nicotine effect.

Central Pathways of MAPKp38 and MAPKJNK Mediate TNFα/iNOS‐Dependent Endotoxic Manifestations of Hypotension and Compromised Heart Rate Variability in Rats

We investigated whether TNFα‐coupled mitogen‐activated protein kinases (MAPKs) in central neurons modulate the early hemodynamic responses evoked by endotoxemia in rats. Changes in blood pressure (BP), heart rate (HR), and HR variability (HRV) caused by i.v. lipopolysaccharide (LPS, 10 mg/kg) were assessed in absence and presence of pharmacologic inhibitors of TNFα, inducible nitric oxide synthase (iNOS), MAPKERK1/2, MAPKp38, or MAPKJNK. Compared with saline, LPS caused significant falls in BP and increases in HR that appeared 30 min post treatment and continued for the following 60 min. The time (standard deviation of beat‐to‐beat intervals, SDNN, and the root mean square of successive beat‐to‐beat differences in R‐R intervals, rMSSD) and frequency domain indices (total power and spectral bands of low and high‐frequency) of HRV were all reduced by LPS. The inhibition of TNFα (pentoxifylline) or iNOS (aminoguanidine) abolished the hemodynamic and HRV effects of LPS. Intracisternal (i.c.) injection of ODQ (guanylate cyclase inhibitor), wortmannin (PI3K inhibitor), SP600125 (MAPKJNK inhibitor) mitigated the LPS‐evoked reductions in BP and HR, but failed to affect the associated decreases in HRV. MAPKp38 inhibition by i.c. SB203580 produced exactly the opposite effects. The reduction in BP or HRV caused by LPS remained unaltered after i.c. PD98059, suggesting no role for MAPKERK1/2 in the LPS responses. Together, the data suggest that central TNFα/iNOS/GC/PI3K pathways linked to MAPKp38 and MAPKJNK are pivotal for provoking the reductions in BP and HRV, respectively, seen during endotoxic shock.

Impairment of HuR-Mediated FOS mRNA Stabilization in Granulocytes From Myelodysplastic Syndrome Patients.

Abstract 2805Infection is a major cause of death in patients with myelodysplastic syndromes (MDS). Although qualitative and quantitative granulocyte defects have been described, the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. Recently, we found that FOS mRNA elevation following translation inhibition was significantly smaller in MDS granulocytes than in healthy granulocytes (Shikama et al, Br J Haematol 154, 525–527, 2011). FOS is an immediate early gene that is rapidly expressed in response to various stimuli and cellular stresses, and possesses an AU-rich element (ARE) in its mRNA 3’UTR, to which mRNA stability-regulating proteins, such as HuR, bind. The aim of this study was to clarify the cause of impaired FOS induction following translation inhibition in MDS.We first confirmed impaired FOS induction in MDS by comparing 17 patients with MDS (refractory anemia) with 17 age-matched healthy controls. Granulocyte fractions, enriched to >90% neutrophils, were cultured with 200 ng/mL of the translation inhibitor emetine at 37 °C for 30 min. FOS mRNA increased 6.2±1.1-fold in controls, vs. 3.5±0.8-fold in MDS (p<0.01). In contrast, when 5 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) or 100 ng/mL lipopolysaccharide (LPS) was added instead of emetine, there were no significant differences in the FOS mRNA elevation between controls and MDS (GM-CSF: 2.4±0.5-fold vs. 2.3±1.3-fold, LPS: 2.2±0.9-fold vs. 2.4±1.1-fold), suggesting that FOS mRNA induction by a translation inhibitor was specifically impaired in MDS granulocytes.To study the mechanisms of FOS mRNA elevation by emetine, we next examined the involvement of (1) MAPK signaling pathways, (2) FOS transcription and mRNA stabilization, and (3) ARE-binding protein HuR, using healthy granulocytes. Inhibition of MAPK p38, which completely blocked the FOS-upregulating effect of LPS, reduced FOS elevation by emetine to nearly 50%. Neither inhibition of JNK nor ERK altered the effects of emetine. Thus, emetine increases FOS mRNA via MAPK p38-dependent and MAPK-independent mechanisms. Emetine treatment for 30 min increased nascent FOS mRNA 3.5±0.8-fold, which was reduced to 1.1±0.8-fold by MAPK p38 inhibition (p<0.01). When transcription was inhibited, FOS mRNA decayed to 19.2±9.1% of the initial level in 45 min. In the presence of emetine, 78.1±23.5% of FOS mRNA remained at 45 min, and was not affected by p38 inhibition. These data indicate that FOS mRNA elevation by emetine results from both upregulation of transcription via MAPK p38 and p38-independent mRNA stabilization. RNA-protein complex immunoprecipitation showed that 2.5±1.2-fold more FOS mRNA 3’UTR coprecipitated with HuR in emetine-treated cells than in untreated cells (p<0.05). GAPDH mRNA 3’UTR without ARE was not detected regardless of emetine treatment. Although HL60 cells treated with HuR and control siRNA showed similar FOS mRNA decay rates in the absence of emetine, emetine treatment led to a significantly lower FOS mRNA at 45 min in HuR-reduced cells (48.0±12.8%) than in control cells (77.4±13.9%, p<0.05). Thus, emetine stabilizes FOS mRNA via upregulated interaction of HuR with FOS mRNA.Finally, we investigated which process was impaired in MDS, p38-mediated FOS transcription, or HuR-mediated mRNA stabilization. The increase of FOS transcription in MDS (2.0±0.3-fold) did not significantly differ from that in controls (2.4±0.3-fold). When FOS transcription was blocked by a MAPK p38 inhibitor, FOS mRNA increase by emetine was significantly smaller in MDS (2.9±0.7-fold) than in controls (4.4±1.5-fold, p<0.05), suggesting that the aberrant FOS induction in MDS was not due to impaired FOS transcription. FOS mRNA, which decayed similarly in MDS and control cells in the absence of emetine, remained 41.9±24.6% and 61.0±19.9% in MDS and control granulocytes, respectively, at 45 min in the presence of emetine (p<0.05).In conclusion, the aberrant FOS mRNA induction by emetine in MDS results from the impairment of HuR-mediated FOS mRNA stabilization. Since nearly 15% of the transcriptome in mammalian cells is targeted by HuR, these results may provide new insights into the functional defects in MDS. Disclosures:No relevant conflicts of interest to declare.

Ethanol transiently suppresses choline-acetyltransferase in basal nucleus of Meynert slices

The cholinergic system plays a major role in learning and cognition and cholinergic neurons appear to be particularly vulnerable to ethanol (EtOH) exposure. There are conflicting results if EtOH directly damages cholinergic neurons. Thus, the aims of the present study were (1) to investigate the effect of different EtOH concentrations on cholinergic neurons in organotypic brain slices of the nucleus basalis of Meynert (nbM) and (2) to study if the most potent cholinotrophic substance nerve growth factor (NGF) or inhibitors of mitogen activated kinase (MAPK) p38- and nitric-oxide synthase (NOS)-pathways may counteract any EtOH effect. Two-week old organotypic rat brain slices of the nbM were exposed to 1–100mM EtOH for 7days with or without drugs and the number of choline-acetyltransferase (ChAT)-positive neurons was counted. Our data show that EtOH significantly reduced the number of ChAT-positive neurons with the most potent effect at a concentration of 50mM EtOH (54±5 neurons per slice, p<0.001), compared to control slices (120±13 neurons per slice). Inhibition of MAPK p38 (SB 203580, 10μM) and NOS (L-thiocitrulline, 10μM) counteracted the EtOH-induced decline of cholinergic neurons and NGF protected cholinergic neurons against the EtOH-induced effect. Withdrawal of EtOH resulted in a reversal of cholinergic neurons to nearly controls. In conclusion, EtOH caused a transient decline of cholinergic neurons, possibly involving MAPK p38- and NOS-pathways suggesting that EtOH does not induce direct cell death, but causes a transient downregulation of the cholinergic key enzyme, possibly reflecting a form of EtOH-associated plasticity.