Activation Of ERK MAPK Research Articles

The Alpha Isoform of Heat Shock Protein 90 and the Co-chaperones p23 and Cdc37 Promote Opioid Anti-nociception in the Brain.

Opioid activation of the mu opioid receptor (MOR) promotes signaling cascades that evoke both analgesic responses to pain and side effects like addiction and dependence. Manipulation of these cascades, such as by biased agonism, has great promise to improve opioid therapy. However, the signaling cascades of the MOR are in general poorly understood, providing few targets for drug development. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel and crucial regulator of opioid anti-nociception in the brain by promoting ERK MAPK activation. In this study, we sought to identify the molecular isoforms and co-chaperones by which Hsp90 carried out this role, which could provide specific targets for future clinical intervention. We used novel selective small molecule inhibitors as well as CRISPR/Cas9 gene editing constructs delivered by the intracerebroventricular (icv) route to the brains of adult CD-1 mice to target Hsp90 isoforms (Hsp90α/β, Grp94) and co-chaperones (p23, Cdc37, Aha1). We found that inhibition of the isoform Hsp90α fully blocked morphine anti-nociception in a model of post-surgical paw incision pain, while blocking ERK and JNK MAPK activation, suggesting Hsp90α as the main regulator of opioid response in the brain. We further found that inhibition of the co-chaperones p23 and Cdc37 blocked morphine anti-nociception, suggesting that these co-chaperones assist Hsp90α in promoting opioid anti-nociception. Lastly, we used cycloheximide treatment in the brain to demonstrate that rapid protein translation within 30 min of opioid treatment is required for Hsp90 regulation of opioid response. Together these studies provide insight into the molecular mechanisms by which Hsp90 promotes opioid anti-nociception. These findings thus both improve our basic science knowledge of MOR signal transduction and could provide future targets for clinical intervention to improve opioid therapy.

Fibroblasts to Keratinocytes Redox Signaling: The Possible Role of ROS in Psoriatic Plaque Formation.

Although the role of reactive oxygen species-mediated (ROS-mediated) signalling in physiologic and pathologic skin conditions has been proven, no data exist on the skin cells ROS-mediated communication. Primary fibroblasts were obtained from lesional and non-lesional skin of psoriatic patients. ROS, superoxide anion, calcium and nitric oxide levels and lipoperoxidation markers and total antioxidant content were measured in fibroblasts. NADPH oxidase activity and NOX1, 2 and 4 expressions were assayed and NOX4 silencing was performed. Fibroblasts and healthy keratinocytes co-culture was performed. MAPK pathways activation was studied in fibroblasts and in co-cultured healthy keratinocytes. Increased intracellular calcium, •NO and ROS levels as well as an enhanced NADPH oxidase 4 (NOX4)–mediated extracellular ROS release was shown in lesional psoriatic vs. control fibroblasts. Upon co-culture with lesional fibroblasts, keratinocytes showed p38 and ERK MAPKs pathways activation, ROS, Ca2+ and •NO increase and cell cycle acceleration. Notably, NOX4 knockdown significantly reduced the observed effects of lesional fibroblasts on keratinocyte cell cycle progression. Co-culture with non-lesional psoriatic and control fibroblasts induced slight cell cycle acceleration, but notable intracellular ROS accumulation and ERK MAPK activation in keratinocytes. Collectively, our data demonstrate that NOX4 expressed in dermal fibroblasts is essential for the redox paracrine regulation of epidermal keratinocytes proliferation.

MAPK-ERK Pathway Activation: The Achilles' Heel of IL-7R‒Induced Steroid Resistance

INTRODUCTION. Mutations in the IL-7R signaling pathway (e.g.IL7Ra, JAK1/3, PTPN11, NF1, STAT5B, N/KRAS or AKT) are associated with steroid resistance and inferior relapse-free survival. These mutant molecules drive strong activation of the JAK-STAT, MAPK-ERK and/or PI3K-AKT signaling pathways. Also, activation of wild type RAS or AKT-reflecting physiological IL7-induced signaling-drives steroid resistance. These observations extend the findings in preB-ALL where RAS mutations were related to steroid resistance. Impaired transcription of pro-apoptotic BIM-an essential direct transcriptional target gene of the glucocorticoid receptor NR3C1 that mediates steroid-induced apoptosis-is observed in various steroid resistant PDX models. AIM. Given the central role of BIM in steroid-induced apoptosis, we investigated how mutations in IL7R or downstream signaling components would affect the function of BIM and other BCL2 protein family members (BCL2, BCLXL and MCL1) in steroid-exposed T-ALL. RESULTS. We generated doxycycline-inducible SUP-T1 and P12 Ichikawa cell line models that can overexpress specific IL-7R signaling mutants. Expression of mutant JAK1, NRAS or IL7Ra molecules results in robust activation of the MAPK-ERK pathway that confers steroid resistance. As a target of NR3C1, BIM transcription was not impaired in these steroid treated cell lines. Therefore, we investigated post-transcriptional mechanisms of steroid resistance. We observed strong phosphorylation of BIM downstream of MAPK-ERK activation under steroid treated and untreated conditions. Exposure to each of three MEK-inhibitors CI1040, Selumetinib or Trametinib abolished not only phospho-ERK but also phosphorylation of BIM. To study the consequences of BIM phosphorylation in relation to binding and inactivation of anti-apoptotic proteins, we immunoprecipitated BIM. Increased BIM levels following steroid exposure was related to increased binding to BCL2, BCLXL and MCL1 in steroid-sensitive control cells. In contrast, activation of mutant JAK1 and NRAS molecules resulted in phosphorylated BIM and reduced binding of BIM to these anti-apoptotic BCL2 family members. This indicates that preventing ERK-mediated phosphorylation of BIM enhances steroid responsiveness. T-ALL patients with activating IL-7R signaling mutations may therefore benefit from treatment combining steroids and MEK inhibitors. Indeed we observed strong synergy between MEK inhibitors and steroids in primary T-ALL patient samples. CONCLUSIONS AND FUTURE PERSPECTIVES. Treatment with MEK-inhibitors Selumetinib or Trametinib was shown effective in reversing steroid resistance, and therefore strongly synergize with steroid treatment. Our data highlights the importance of the dynamic interplay between BIM and anti-apoptotic BCL2 family members in MAPK-ERK-driven steroid resistance. Therefore, we are exploring whether MEK inhibitors (that prevent phosphorylation and inactivation of BIM) combined with BH3-mimetics (that block anti-apoptotic family members) will further enhance restoration of steroid responsiveness in IL-7R signaling mutant T-ALL patients. As physiological IL7-induced signaling is sufficient to raise cellular steroid resistance, these inhibitors could be applied for all IL-7R signaling-dependent T-ALL patients, also in the absence of IL-7R signaling mutations. Refractory/relapse T-ALL patients are now eligible for the current phase 1/2 MEK-inhibitor‒dexamethasone (SeluDex) trial. We are using mass-spectrometry to determine the exact phospho-sites in BIM that drives steroid resistance that can serve as a valuable biomarker. Disclosures No relevant conflicts of interest to declare.

S14-09 SESSION 14: FACIOCRANIOSYNOSTOSIS – PART III MITIGATION OF SHP2 ACTIVATION PREVENTS FGFR2 SIGNALING INDUCED CRANIOSYNOSTOSIS

Introduction: Activating-dominant mutations in the fibroblast growth factor receptor IIIc splice variant gene (Fgfr2c) cause premature cranial suture fusion (craniosynostosis), which can restrict growth of the craniofacial complex. Fgfr2c signaling is mediated via the docking protein Frs2a, which interacts with both Shp2, a tyrosine phosphatase, and Grb2, an adapter protein, to induce ERK-MAPK activation. The role of both Shp2 and Grb2 in craniosynostosis and craniofacial growth remains unknown; however, it is known that gain-of-function Fgfr2c mutant mice demonstrate hyperactivation of the ERK-MAPK signaling cascade and osteoblast activity. The objective of the current study is to determine the role Shp2 and Grb2 may play in Fgfr2-induced craniosynostosis and craniofacial growth. Methods: Gain-of-function Fgfr2c mice were crossed with mice deficient in two Shp2- or four Grb2-specific tyrosine phosphorylation sites within Frs2a. Skull morphology and sutures were analyzed grossly, histologically, and by micro-Computed Tomography. Coronal sutures were analyzed for ERK protein activation and whole calvaria were cultured and analyzed for alkaline phosphatase activity. Fluorescence-lifetime imaging microscopy – Fluorescence Resonance Energy Transfer (FLIM-FRET) was performed to study the interactions between FRS2a, Shp2, and Grb2 within WT or gain-of-function Fgfr2c mutant HEK293T cells. Analysis of Variance was used to compare three or more groups. An observed P value ≤ 0.05 was considered statistically significant. Results: Reduction of Shp2 activation prevents coronal suture fusion and restores normal craniofacial growth (P < 0.05), ERK-MAPK signaling (P < 0.05), and osteoblast activity (P < 0.05) in a mouse model of Fgfr2c-induced craniosynostosis via an Frs2a-dependent mechanism. Complete uncoupling of Grb2 from Frs2a resulted in only partial rescue of normal suture formation and growth. FLIM-FRET analysis revealed a complex interplay between FRS2a, Shp2, and Grb2 in the setting of dysregulated Fgfr2c signaling. Conclusion: This study provides mechanistic insight into craniofacial development and identifies Shp2 as a key regulator of craniofacial growth and development and highlights the therapeutic potential of Shp2 modulation for the treatment of Fgfr2c-related disorders.

GluN2A-NMDA receptor–mediated sustained Ca2+ influx leads to homocysteine-induced neuronal cell death

Homocysteine, a metabolite of the methionine cycle, is a known agonist of N-methyl-d-aspartate receptor (NMDAR), a glutamate receptor subtype and is involved in NMDAR-mediated neurotoxicity. Our previous findings have shown that homocysteine-induced, NMDAR-mediated neurotoxicity is facilitated by a sustained increase in phosphorylation and activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK MAPK). In the current study, we investigated the role GluN1/GluN2A-containing functional NMDAR (GluN2A-NMDAR) and GluN1/GluN2B-containing functional NMDAR (GluN2B-NMDAR) in homocysteine-induced neurotoxicity. Our findings revealed that exposing primary cortical neuronal cultures to homocysteine leads to a sustained low-level increase in intracellular Ca2+ We also showed that pharmacological inhibition of GluN2A-NMDAR or genetic deletion of the GluN2A subunit attenuates homocysteine-induced increase in intracellular Ca2+ Our results further established the role of GluN2A-NMDAR in homocysteine-mediated sustained ERK MAPK phosphorylation and neuronal cell death. Of note, the preferential role of GluN2A-NMDAR in homocysteine-induced neurotoxicity was distinctly different from glutamate-NMDAR-induced excitotoxic cell death that involves overactivation of GluN2B-NMDAR and is independent of ERK MAPK activation. These findings indicate a critical role of GluN2A-NMDAR-mediated signaling in homocysteine-induced neurotoxicity.

ZAP70 deficiency promotes reverse cholesterol transport through MAPK/ERK pathway in Jurkat cell

BackgroundLots of studies have demonstrated that immune cells could regulate reverse cholesterol transport (RCT). However, neither T cell receptor (TCR) signalling nor Zeta-chain associated protein 70 (ZAP70) have been demonstrated to be associated with RCT. To investigate this association, we used a ZAP70-deficient Jurkat-derived mutant, P116 cell line, to detect the effect of ZAP70 on RCT and inflammatory response. ZAP70 deficiency improved cholesterol efflux capacity by 14%. Meanwhile, mRNA and proteins expression of RCT regulatory proteins such as ABCA1, ABCG1 and SR-BI were increased in P116 cells. ZAP70-deficiency had no influence on LXR-α and PPAR-γ. Regarding the inflammatory response, the mRNA expression and secretion of pro-atherosclerotic cytokines, TNF-α, IFN-γ, IL-2 and IL-6, were significantly decreased in the ZAP70-deficient cell line. Activation of MAP kinases cascades, as determined by of ERK, JNK and p38 MAPK phosphorylation, were found to be inhibited in the absence of ZAP70. Specific inhibition of ERK, JNK and p38 MAPK activity was also found to decreased TNF-α, IFN-γ, and IL-6 secretion. However, only the ERK inhibition was observed to reduce IL-2 secretion, improve cholesterol efflux capacity and increase expression of ABCA1, ABCG1 and SR-BI without increasing LXR-α and PPAR-γ. Using ChIP assay to detect the binding of LXR-α to LXRE, which promotes the expression of ABCG1, we found that inhibiting ERK improved binding without increasing LXR-α levels. Thus, we speculate that ZAP70-deficiency may improve RCT and decrease the inflammatory response of T cells. Furthermore, these effects are probably achieved via ERK signalling pathway.

Targeting B7-H1 (PD-L1) sensitizes cancer cells to chemotherapy

Development of resistance to chemotherapy is a major obstacle in extending the survival of patients with cancer. Although originally defined as an immune checkpoint molecule, B7-H1 (also named as PD-L1 or CD274) was found to play a role in cancer chemoresistance; however, the underlying mechanism of action of B7-H1 in regulation of chemotherapy sensitivity remains unclear in cancer cells. Here we show that development of chemoresistance depends on an increased activation of ERK in cancer cells overexpressing B7-H1. Conversely, B7-H1 knockout (KO) by CRISPR/Cas9 renders human cancer cells susceptible to chemotherapy in a cell-context dependent manner through a reduced activation of p38 MAPK. B7-H1 was found to associate with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and this association promoted or maintained the activation of ERK or p38 MAPK in cancer cells. Importantly, we found that targeting B7-H1 by anti-B7-H1 monoclonal antibody (H1A) increased the sensitivity of human triple negative breast cancer cells to cisplatin therapy in vivo. Our results suggest that targeting B7-H1 by an antibody capable of disrupting B7-H1 signals may be a new approach to sensitize cancer cells to chemotherapy.

MUC5AC overexpression by organic extract of SRM 1648 urban particulate matter via ERK, JNK and p38 MAPK activation in Calu-3 human airway epithelial cells

MUC5AC overexpression by organic extract of SRM 1648 urban particulate matter via ERK, JNK and p38 MAPK activation in Calu-3 human airway epithelial cells

Alkyl-imino sugars inhibit the pro-oncogenic ion channel function of human papillomavirus (HPV) E5

Despite the availability of prophylactic vaccines the burden of human papillomavirus (HPV) associated malignancy remains high and there is a need to develop additional therapeutic strategies to complement vaccination. We have previously shown that the poorly characterised E5 oncoprotein forms a virus-coded ion channel or viroporin that was sensitive to the amantadine derivative rimantadine. We now demonstrate that alkylated imino sugars, which have antiviral activity against a number of viruses, inhibit E5 channel activity in vitro. Using molecular modelling we predict that imino sugars intercalate between E5 protomers to prevent channel oligomerisation. We explored the ability of these viroporin inhibitors to block E5-mediated activation of mitogenic signalling in keratinocytes. Treatment with either rimantadine or imino sugars prevented ERK-MAPK phosphorylation and reduced cyclin B1 expression in cells expressing E5 from a number of high-risk HPV types. Moreover, viroporin inhibitors also reduced ERK-MAPK activation and cyclin B1 expression in differentiating primary human keratinocytes containing high-risk HPV18. These observations provide evidence of a key role for E5 viroporin function during the HPV life cycle. Viroporin inhibitors could be utilised for stratified treatment of HPV associated tumours prior to virus integration, or as true antiviral therapies to eliminate virus prior to malignant transformation.

Exendin-4, a Glucagon-Like Peptide-1 Receptor Agonist, Attenuates Prostate Cancer Cell Proliferation via Phosphorylation of MKP-1

Pleiotropic benefits of incretin therapy beyond glycemic control have been reported and receiving much attention. Cancer is one of major causes of death in patients with type 2 diabetes. We have previously reported Exendin-4(Ex-4), a glucagon-like peptide-1 receptor agonist, attenuates prostate cancer growth by the inhibition of ERK-MAPK phosphorylation (Diabetes 2014). On the other hand, it is well known that MAPK is inactivated and tightly regulated by MAPK phosphatase (MKP) family. Then, we next examined whether MKP could be involved in the anti-proliferative effect of Ex-4 in the prostate cancer cell. In a human prostate cancer cell line, LNCaP cell, MKP-1 was abundantly expressed. Knocking down of MKP-1 using siRNA abolished Ex-4 induced-reduction of LNCaP cell number in growth curve. In addition, Ex-4 treatment significantly increased MKP-1 mRNA expression. To activate its phosphatase activity, Ser359 in the C terminus of MKP-1 is specifically phosphorylated. Ex-4 significantly phosphorylated Ser359 of MKP-1along with time course of dephosphorylating ERK-MAPK. Using Immunoprecipitation assay, we further confirmed that Ex-4 exactly phosphorylated Ser359 of MKP-1. In addition, if we transiently transfected GFP conjugated MKP-1 into LNCaP cells, nuclear translocation of MKP-1 induced by Ex-4 treatment was observed in early phase stimulation. These data suggest that Ex-4 attenuates prostate cancer cell proliferation via phosphorylation of MKP-1 followed by inhibition of ERK-MAPK activation. Disclosure T. Kawanami: None. T. Nomiyama: Research Support; Self; MSD K.K., Sanofi-Aventis, Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan. Y. Hamaguchi: None. T. Tanaka: None. T. Yanase: Research Support; Self; MSD K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.

Heat Shock Protein 90α, Assisted by Co‐Chaperones p23 and Cdc37, Promotes Opioid Anti‐Nociception in the Brain via Promoting ERK MAPK Signaling

Reducing opioid side effects like constipation and addiction has been a strong focus for improving opioid therapy in treating acute and chronic pain. One such approach includes manipulation of mu opioid receptor (MOR) signaling cascades, such as βarrestin2. However, the MOR signaling complex and mechanisms of signaling regulation are poorly understood. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel MOR signaling regulator, which could be a target for improving the therapeutic profile of opioids. We found that intracerebroventricular (icv) 17‐AAG, a well‐established Hsp90 inhibitor, strongly blocked or abolished morphine anti‐nociception in paw incision and HIV neuropathic pain in mice. The current study was designed to identify molecular mechanisms of this regulation, including which isoform(s) and/or co‐chaperone(s) are involved in the regulatory effect of Hsp90 on opioid signaling. Male and female CD‐1 mice were treated with selective inhibitors for different Hsp90 isoforms (KU‐A, KUNB106, KUNG65) and co‐chaperones (KU177, gedunin, celastrol) via icv injection. We found that an Hsp90α selective inhibitor blocked morphine anti‐nociception by ~ 80% in the paw incision model, however, inhibitors for Hsp90β and Grp94 had no impact on morphine anti‐nociception. Knockdown of Hsp90α in the brain by CRISPR‐Cas9 also showed strong inhibition of morphine anti‐nociception in paw incision, confirming the results. Further, inhibition of the co‐chaperones p23 and Cdc37, but not Aha1, showed similar effects as Hsp90α inhibition in the paw incision model. To determine the molecular mechanism of these activities, we found that Hsp90α inhibitor treatment blocked ERK and JNK MAPK activation induced by the selective MOR agonist DAMGO without changing the expression of Hsp70 in mouse periaqueductal grey (PAG). Treatment with the ERK MAPK inhibitor U0126 recapitulated our results of Hsp90 inhibition on paw incision, HIV neuropathic, and tail flick pain, suggesting that ERK MAPK regulation is the means by which Hsp90α regulates morphine anti‐nociception. Identification of the isoforms and co‐chaperones involved in Hsp90 regulation of ERK and opioid anti‐nociception may permit more selective manipulation of the MOR signaling machinery to improve opioid efficacy and reduce side effects.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Heat Shock Protein 90 in the Spinal Cord Strongly Promotes Morphine Anti Nociception by Increasing ERK MAPK Activation

Hundreds of millions of individuals suffer from acute and chronic pain in the United States alone. The current gold standard of treatment for patients with moderate to severe pain is the use of opioid analgesics. Although the efficacy of these medications is often superior to other options in the clinic, their use is significantly limited due to their side effects and potential for abuse. Recent progress in the research of opioid receptor signaling has shown that manipulation of opioid signaling by biased agonists or other means can improve the therapeutic profile of opioids; however, the field is limited by a lack of understanding of the opioid signaling complex. We have previously shown that inhibition of the chaperone protein Heat shock protein 90 (Hsp90) in the brain strongly blocks opioid anti‐nociception by blocking ERK activation. Here we demonstrate a reversed effect in the spinal cord in that intrathecal (i.t.) injections of the Hsp90 inhibitors 17‐AAG or KU‐32 in CD‐1 mice strongly increase morphine anti‐nociception in paw incision and tail flick pain. Seeking a mechanism for these changes, we found that i.t. Hsp90 inhibition strongly increased ERK activation in the spinal cord in response to opioids, which is opposite of what is observed in the brain, and also implies that Hsp90 acts to repress opioid‐stimulated ERK signaling in the spinal cord. By combining Hsp90 inhibition with the ERK inhibitor U0126 in the spinal cord, we found that ERK inhibition fully reversed the effects of Hsp90 inhibition while having no effect on its own, supporting the hypothesis that increased ERK signaling in spinal cord is responsible for the effects of Hsp90 inhibition. We also performed experiments with systemic and combined i.c.v. and i.t. Hsp90 inhibitor to demonstrate that Hsp90 inhibition in the brain dominates over other circuits when all are inhibited. This data implies that signaling localization within the pain circuit could be key for the targeting of proteins to modify the therapeutic index of opioid analgesics like morphine. These results also suggest that spinal Hsp90 inhibitors could be used as an opioid dose‐reduction strategy which could preserve analgesic efficacy while reducing side effects.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impaired Ca2+ handling in resistance arteries from genetically obese Zucker rats: Role of the PI3K, ERK1/2 and PKC signaling pathways

The impact of obesity on vascular smooth muscle (VSM) Ca2+ handling and vasoconstriction, and its regulation by the phosphatidylinositol 3-kinase (PI3K), mitogen activated protein kinase (MAPK) and protein kinase C (PKC) were assessed in mesenteric arteries (MA) from obese Zucker rats (OZR). Simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and tension were performed in MA from OZR and compared to lean Zucker rats (LZR), and the effects of selective inhibitors of PI3K, ERK-MAPK kinase and PKC were assessed on the functional responses of VSM voltage-dependent L-type Ca2+ channels (CaV1.2). Increases in [Ca2+]i induced by α1-adrenoceptor activation and high K+ depolarization were not different in arteries from LZR and OZR although vasoconstriction was enhanced in OZR. Blockade of the ryanodine receptor (RyR) and of Ca2+ release from the sarcoplasmic reticulum (SR) markedly reduced depolarization-induced Ca2+ responses in arteries from lean but not obese rats, suggesting impaired Ca2+-induced Ca2+ release (CICR) from SR in arteries from OZR. Enhanced Ca2+ influx after treatment with ryanodine was abolished by nifedipine and coupled to up-regulation of CaV1.2 channels in arteries from OZR. Increased activation of ERK-MAPK and up-regulation of PI3Kδ, PKCβ and δ isoforms were associated to larger inhibitory effects of PI3K, MAPK and PKC blockers on VSM L-type channel Ca2+ entry in OZR. Changes in arterial Ca2+ handling in obesity involve SR Ca2+ store dysfunction and enhanced VSM Ca2+ entry through L-type channels, linked to a compensatory up-regulation of CaV1.2 proteins and increased activity of the ERK-MAPK, PI3Kδ and PKCβ and δ, signaling pathways.

K63-Linked Polyubiquitination on TRAF6 Regulates LPS-Mediated MAPK Activation, Cytokine Production, and Bacterial Clearance in Toll-Like Receptor 7/8 Primed Murine Macrophages.

Post viral infection bacterial pneumonia is a major cause of morbidity and mortality associated with both seasonal and pandemic influenza virus illness. Despite much efforts put into the discovery of mechanisms of post viral–bacterial infections and their complications in recent years, the molecular mechanisms underlying the increased susceptibility to bacterial infection remain poorly understood. In this study, we focused on the pathways regulating immune responses in murine macrophages and modeled post viral–bacterial infections through pretreatment of bone marrow-derived macrophages (BMDMs) with a toll-like receptor (TLR) 7/8 ligand (R848) and subsequent challenge with TLR2/4 agonists to mimic bacterial infection. We found R848-primed BMDMs upon subsequent exposure to TLR2/4 ligands respond with enhanced inflammatory cytokine production, especially IL-6 and TNF-α. The enhanced cytokine production in R848-primed BMDMs in response to TLR2/4 was due to increased TGF-β-activated kinase (TAK) 1 phosphorylation with subsequent activation of ERK and p38 MAPKs. Furthermore, we identified that R848 priming leads to increased K63-linked polyubiquitination on TRAF6. K63-linked polyubiquitination on TRAF6 is a signal leading to enhanced activation of downstream pathways including TAK1. Importantly, R848-primed BMDMs infected with live bacteria exhibited decreased bacterial clearance. Small-molecule enhancer of rapamycin 3, an ubiquitin ligase inhibitor reversed the K63-linked polyubiquitination on TRAF6 in R848-primed BMDMs and subsequently decreased TAK1 and MAPK phosphorylation, and cytokine production as well as reversed the decreased bacterial clearance capacity of BMDMs. Our study may provide a novel molecular target to alleviate post viral–bacterial infections.

(312) - Heat shock protein 90 promotes opioid anti-nociception in the brain and represses opioid anti-nociception in the spinal cord through the differential regulation of ERK MAPK signaling

Opioid drugs are efficacious in treating moderate to severe acute and chronic pain, but are limited by serious side effects. Recent efforts to create biased opioids without these side effects by selectively engaging downstream signal transduction (e.g. βarrestin2) has been limited by our lack of knowledge of the mu opioid receptor (MOR) signaling complex. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel MOR signaling regulator. We found that intracerebroventricular (icv) 17-AAG, a well-established Hsp90 inhibitor, strongly blocked or abolished morphine anti-nociception in paw incision and HIV neuropathy pain in mice. In our current work, we've extended from these findings to examine the role of Hsp90 in the spinal cord. In contrast to our findings in the brain, we found that intrathecal (it) .5 nmol 17-AAG for 24 hours in CD-1 mice caused strongly increased morphine anti-nociception in tail flick and paw incision pain (80.7–88.6% increase). This was confirmed using the alternate Hsp90 inhibitor KU-32 as well as Rotarod testing, ruling out off-target effects. We examined ERK MAPK signaling in the brain and spinal cord in response to opioids after 17-AAG inhibition, and found that ERK MAPK activation was lost in the brain, but potentiated in the spinal cord. By using icv and it administration of 5 µg U0126, a selective ERK inhibitor, we demonstrated that the effects of Hsp90 inhibition in the brain and spinal cord could be completely explained by ERK signaling changes. By using selective inhibitors, we further found that the molecular isoform Hsp90α and the co-chaperones p23 and Cdc37 (but not Aha1) were responsible for the signaling and anti-nociception changes observed in the brain. Overall, these results demonstrate different regional effects of Hsp90 and ERK signaling that have a strong impact on opioid anti-nociception, which could be used to improve opioid drug discovery.

Selective Androgen Receptor Modulator S42 Suppresses Prostate Cancer Cell Proliferation.

We previously identified the selective androgen receptor (AR) modulator S42, which does not stimulate prostate growth but has a beneficial effect on lipid metabolism. In the prostate cancer (PC) cell line LNCaP, S42 did not induce AR transactivation but antagonized 5α-dihydrotestosterone (DHT)‒induced AR activation. Next, we investigated whether S42 suppresses the growth of PC cell lines. Basal growth of LNCaP cells was significantly suppressed by treatment with S42 compared with vehicle, as determined by cell counting and 5-bromo-2'-deoxyuridine assays. The suppressive effect of S42 on cell growth was evident in the AR-positive PC cells LNCaP and 22Rv1 and was slightly observed even in the AR-negative PC-3 cells. However, S42 did not induce apoptosis as determined by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. S42 had an even greater suppressive effect on DHT-dependent LNCaP cell proliferation than on basal proliferation (P < 0.05). DHT treatment increased the expression of phosphorylated extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK), a major signaling molecule for PC proliferation, and this was significantly inhibited by S42. DHT also significantly upregulated AR, insulinlike growth factor-1 receptor (IGF-1R), and insulin receptor (IR)-β protein levels, which were similarly reduced by S42 treatment. Importantly, S42 administration to mice attenuated the growth of LNCaP tumors and reduced tumor expression of the prostate-specific antigen, P504S, Ki67, and phosphorylated ERK-MAPK. These data suggest that S42 attenuates LNCaP tumor growth not by inducing apoptosis but by inhibiting the expression of proliferation-related receptors, including IGF-1R, IR, and AR, and by suppressing ERK-MAPK activation. S42 may thus be a feasible candidate for PC treatment.

Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis.

VEGF binding to VEGFR2 leads to VEGFR2 endocytosis and downstream signaling activation to promote angiogenesis.Methods: Using genetic strategies, we tested the requirement of α subunits of heterotrimeric G proteins (Gαi1/3) in the process.Results: Gαi1/3 are located in the VEGFR2 endocytosis complex (VEGFR2-Ephrin-B2-Dab2-PAR-3), where they are required for VEGFR2 endocytosis and downstream signaling transduction. Gαi1/3 knockdown, knockout or dominant negative mutation inhibited VEGF-induced VEGFR2 endocytosis, and downstream Akt-mTOR and Erk-MAPK activation. Functional studies show that Gαi1/3 shRNA inhibited VEGF-induced proliferation, invasion, migration and vessel-like tube formation of HUVECs. In vivo, Gαi1/3 shRNA lentivirus inhibited alkali burn-induced neovascularization in mouse cornea. Further, oxygen-induced retinopathy (OIR)-induced retinal neovascularization was inhibited by intravitreal injection of Gαi1/3 shRNA lentivirus. Moreover, in vivo angiogenesis by alkali burn and OIR was significantly attenuated in Gαi1/3 double knockout mice. Significantly, Gαi1/3 proteins are upregulated in proliferative retinal tissues of proliferative diabetic retinopathy (PDR) patients.Conclusion: These results provide mechanistic insights into the critical role played by Gαi1/3 proteins in VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis.

Characterisation of cellular effects of Burkholderia pseudomallei cycle inhibiting factor (Cif).

ABSTRACTCycle inhibiting factors (Cifs) are type III secretion system effectors produced by some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Through their deamidase activity, Cifs inhibit the activity of Cullin RING E3 ubiquitin ligases (CRL). CRL inhibition induces the accumulation of cell cycle inhibitors p21 and p27, thereby leading to host cell cycle arrest. However, whether Cif exerts additional effects on host cells that are important in bacterial pathogenesis is currently poorly understood. In this study, we found that Cif exerts a bimodal effect on NF-κB signalling. Cif increases basal NF-κB activity. This effect is dependent on Cif-mediated activation of ERK MAPK. On the other hand, Cif inhibits NF-κB activation by TNFα and Burkholderia thailandensis infection. This inhibitory effect on NF-κB activity is partially mediated by Cif-dependent inhibition of CRLs. We also found that Cif only has a modest effect in stimulating the intracellular replication of the B. pseudomallei surrogate, B. thailandensis. The observed Cif-dependent stimulation of B. thailandensis intracellular replication was not, or was only partially, due to CRL inhibition. Furthermore, the increased B. thailandensis replication induced by Cif was independent of ERK MAPK activation. Our findings suggest that Cif likely exerts additional cellular effects through novel targets.

In1-ghrelin splicing variant is associated with reduced disease-free survival of breast cancer patients and increases malignancy of breast cancer cells lines.

Ghrelin gene generates several variants that regulate multiple pathophysiological functions, including tumor-related processes. In1-ghrelin is a splicing variant that was previously shown to be overexpressed in breast cancer (BCa), where it correlated with proliferation markers; however, its possible association with clinical outcome of BCa patients and underlying mechanisms are still unknown. To address this issue, expression levels and clinical associations of In1-ghrelin were analyzed in a cohort of 117 BCa samples. Additionally, a battery of cellular and molecular assays was implemented using two BCa cell lines (MCF-7 and MDA-MB-231), wherein the role of In1-ghrelin on proliferation, migration, dedifferentiation and signaling pathways was explored. The results generated revealed that high expression of In1-ghrelin in BCa samples was associated with lymph node metastasis and reduced disease-free survival. Indeed, In1-ghrelin overexpression stimulated proliferation and migration in MCF-7 and MDA-MB-231 cells. Similar results were found by treating MDA-MB-231 and MCF-7 with In1-ghrelin-derived peptides. Conversely, In1-ghrelin silencing decreased proliferation and migration capacities of MDA-MB-231. Furthermore, In1-ghrelin (but not ghrelin) overexpression increased the capacity to form mammospheres in both cell lines. These effects could be associated with activation of MAPK-ERK, Jag1/Notch, Wnt/β-catenin and/or TGF-β1 pathways. Altogether, our data indicate that In1-ghrelin could play relevant functional roles in the regulation of BCa development and progression and may provide insights to identify novel biomarkers and new therapeutic approaches for this pathology.

Nelumbo nucifera leaf polyphenol extract inhibits breast cancer cells metastasis in vitro and in vivo through PKCα targeting

Nelumbo nucifera Gaertn. has long been used in food and traditional herbal medicine in Asia. It exhibits several biological properties including antitumor, antiangiogenic, antiobesity, and antioxidant properties. However, the active components and mechanisms underlying the effects of N. nucifera leaf extract (NLE) on breast tumor migration remain undefined. We found that NLE and N. nucifera leaf polyphenol extract (NLPE) significantly attenuated migration in MDA-MB-231 and 4T-1 breast cancer cells. The treatment of cells with NLE and NLPE suppressed the expression of RhoA, Rac1, and Cdc42. Furthermore, the activation of ERK and p38 MAPK was inhibited by NLE and NLPE. Notably, NLE and NLPE treatment significantly reduced the activation of protein kinase C α and the lung metastasis of breast cancer in vivo. Overall, our findings illustrate the roles of NLE and NLPE in breast cancer cell migration, suggesting that NLE and NLPE are potential chemopreventive agents for breast cancer metastasis.