AT2R Activation Prevents Microglia Pro-inflammatory Activation in a NOX-Dependent Manner: Inhibition of PKC Activation and p47phox Phosphorylation by PP2A

Abstract

Microglia-induced reactive oxygen species (ROS) production and inflammation play an imperative role in neurodegenerative diseases like Alzheimer’s disease (AD) and Parkinson’s disease (PD). It has been established that angiotensin II type-2 receptor (AT2R) activation is neuroprotective in central nervous system diseases like stroke and AD. However, the involvement of AT2R in NADPH oxidase (NOX)-mediated microglia activation is still elusive. Therefore, the present study investigated the role of AT2R in angiotensin II (Ang II) or Phorbol 12-myristate 13-acetate (PMA)-induced microglia activation in BV2 cells, primary microglia, p47phox knockout (p47KO) microglia, and in vivo. Treatment of microglia with Ang II or PMA induced a significant ROS generation and promoted pro-inflammatory microglia in a NOX-dependent manner. In contrast, AT2R activation by CGP42112A (CGP) inhibited NOX activation, ROS production, and pro-inflammatory microglia activation, while promoting the immunoregulatory microglia. This inhibitory effect of AT2R on NOX and pro-inflammatory activation was attenuated by AT2R antagonist, PD123319. Essentially, NOX inhibition (by DPI) or scavenging cellular ROS (by NAC) or p47KO microglia were immune to Ang II- or PMA-induced pro-inflammatory microglia activation. Mechanistically, AT2R, via activation of protein phosphatase-2A (PP2A), prevented the Ang II- or PMA-induced protein kinase C (PKC) activation and phosphorylation of p47phox, an effect that was reversed by the addition of PP2A inhibitor, Okadaic acid (OA). Importantly, PKC inhibitor, Rottlerin, inhibited the Ang II- or PMA-induced p47phox phosphorylation and ROS generation to the similar extent as AT2R activation. In addition, AT2R activation or p47KO prevented ROS production, pro-inflammatory microglial activation, and sickness behavior in mice model of neuroinflammation. Therefore, the present findings suggested that AT2R, via PP2A-mediated inhibition of PKC, prevents the NOX activation, ROS generation, and subsequent pro-inflammatory activation of microglia.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

References

  1. 1.

    Perry VH, Nicoll JA, Holmes C (2010) Microglia in neurodegenerative disease. Nat Rev Neurol 6:193–201

    Article  Google Scholar 

  2. 2.

    Yenari MA, Xu L, Tang XN, Qiao Y, Giffard RG (2006) Microglia potentiate damage to blood-brain barrier constituents: improvement by minocycline in vivo and in vitro. Stroke 37:1087–1093

    Article  Google Scholar 

  3. 3.

    Saijo K, Glass CK (2011) Microglial cell origin and phenotypes in health and disease. Nat Rev Immunol 11:775–787

    CAS  Article  Google Scholar 

  4. 4.

    Rojo AI, McBean G, Cindric M, Egea J, Lopez MG, Rada P, Zarkovic N, Cuadrado A (2014) Redox control of microglial function: molecular mechanisms and functional significance. Antioxid Redox Signal 21:1766–1801

    CAS  Article  Google Scholar 

  5. 5.

    Salemi J, Obregon DF, Cobb A, Reed S, Sadic E, Jin J, Fernandez F, Tan J et al (2011) Flipping the switches: CD40 and CD45 modulation of microglial activation states in HIV associated dementia (HAD). Mol Neurodegener 6:3

    CAS  Article  Google Scholar 

  6. 6.

    Hu X, Leak RK, Shi Y, Suenaga J, Gao Y, Zheng P, Chen J (2015) Microglial and macrophage polarization-new prospects for brain repair. Nat Rev Neurol 11:56–64

    Article  Google Scholar 

  7. 7.

    Andersen JK (2004) Oxidative stress in neurodegeneration: cause or consequence? Nat Med 10(Suppl):S18–S25

    Article  Google Scholar 

  8. 8.

    Mariani E, Polidori MC, Cherubini A, Mecocci P (2005) Oxidative stress in brain aging, neurodegenerative and vascular diseases: an overview. J Chromatogr B Analyt Technol Biomed Life Sci 827:65–75

    CAS  Article  Google Scholar 

  9. 9.

    Nakagawa Y, Chiba K (2014) Role of microglial m1/m2 polarization in relapse and remission of psychiatric disorders and diseases. Pharmaceuticals (Basel) 7:1028–1048

    CAS  Article  Google Scholar 

  10. 10.

    Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245–313

    CAS  Article  Google Scholar 

  11. 11.

    Choi SH, Aid S, Kim HW, Jackson SH, Bosetti F (2012) Inhibition of NADPH oxidase promotes alternative and anti-inflammatory microglial activation during neuroinflammation. J Neurochem 120:292–301

    CAS  Article  Google Scholar 

  12. 12.

    Egger T, Schuligoi R, Wintersperger A, Amann R, Malle E, Sattler W (2003) Vitamin E (alpha-tocopherol) attenuates cyclo-oxygenase 2 transcription and synthesis in immortalized murine BV-2 microglia. Biochem J 370(Pt 2):459–467. https://doi.org/10.1042/BJ20021358

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Rodriguez-Pallares J, Rey P, Parga JA, Munoz A, Guerra MJ, Labandeira-Garcia JL (2008) Brain angiotensin enhances dopaminergic cell death via microglial activation and NADPH-derived ROS. Neurobiol Dis 31:58–73

    CAS  Article  Google Scholar 

  14. 14.

    Rodriguez-Perez AI, Borrajo A, Rodriguez-Pallares J, Guerra MJ, Labandeira-Garcia JL (2015) Interaction between NADPH-oxidase and rho-kinase in angiotensin II-induced microglial activation. Glia 63:466–482

    Article  Google Scholar 

  15. 15.

    Liao B, Zhao W, Beers DR, Henkel JS, Appel SH (2012) Transformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS. Exp Neurol 237:147–152

    CAS  Article  Google Scholar 

  16. 16.

    Saavedra JM (2012) Angiotensin II AT(1) receptor blockers as treatments for inflammatory brain disorders. Clin Sci (Lond) 123:567–590

    CAS  Article  Google Scholar 

  17. 17.

    Tota S, Kamat PK, Saxena G, Hanif K, Najmi AK, Nath C (2012) Central angiotensin converting enzyme facilitates memory impairment in intracerebroventricular streptozotocin treated rats. Behav Brain Res 226:317–330

    CAS  Article  Google Scholar 

  18. 18.

    Grammatopoulos TN, Jones SM, Ahmadi FA, Hoover BR, Snell LD, Skoch J, Jhaveri VV, Poczobutt AM et al (2007) Angiotensin type 1 receptor antagonist losartan, reduces MPTP-induced degeneration of dopaminergic neurons in substantia nigra. Mol Neurodegener 2:1

    Article  Google Scholar 

  19. 19.

    Bhat SA, Goel R, Shukla R, Hanif K (2016) Angiotensin receptor blockade modulates NFκB and STAT3 signaling and inhibits glial activation and neuroinflammation better than angiotensin-converting enzyme inhibition. Mol Neurobiol 53(10):6950–6967

    CAS  Article  Google Scholar 

  20. 20.

    Yamamoto S, Yancey PG, Zuo Y, Ma L-J, Kaseda R, Fogo AB, Ichikawa I, Linton MF et al (2011) Macrophage polarization by angiotensin II-type 1 receptor aggravates renal injury-acceleration of atherosclerosis. Arterioscler Thromb Vasc Biol 31(12):2856–2864. https://doi.org/10.1161/ATVBAHA.111.237198

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Dikalov SI, Nazarewicz RR (2013) Angiotensin II-induced production of mitochondrial reactive oxygen species: potential mechanisms and relevance for cardiovascular disease. Antioxid Redox Signal 19:1085–1094

    CAS  Article  Google Scholar 

  22. 22.

    McCarthy CA, Facey LJ, Widdop RE (2014) The protective arms of the renin-angiontensin system in stroke. Curr Hypertens Rep 16:440

    Article  Google Scholar 

  23. 23.

    McCarthy CA, Vinh A, Callaway JK, Widdop RE (2009) Angiotensin AT2 receptor stimulation causes neuroprotection in a conscious rat model of stroke. Stroke 40:1482–1489

    CAS  Article  Google Scholar 

  24. 24.

    Labandeira-Garcia JL, Rodríguez-Perez AI, Garrido-Gil P, Rodriguez-Pallares J, Lanciego JL, Guerra MJ (2017) Brain renin-angiotensin system and microglial polarization: implications for aging and neurodegeneration. Front Aging Neurosci 9:129. https://doi.org/10.3389/fnagi.2017.00129

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Guimond MO, Gallo-Payet N (2012) How does angiotensin AT(2) receptor activation help neuronal differentiation and improve neuronal pathological situations? Front Endocrinol (Lausanne) 3:164

    CAS  Article  Google Scholar 

  26. 26.

    Iwai M, Liu HW, Chen R, Ide A, Okamoto S, Hata R, Sakanaka M, Shiuchi T et al (2004) Possible inhibition of focal cerebral ischemia by angiotensin II type 2 receptor stimulation. Circulation 110:843–848

    CAS  Article  Google Scholar 

  27. 27.

    Reinecke K, Lucius R, Reinecke A, Rickert U, Herdegen T, Unger T (2003) Angiotensin II accelerates functional recovery in the rat sciatic nerve in vivo: role of the AT2 receptor and the transcription factor NF-kappaB. FASEB J 17:2094–2096

    CAS  Article  Google Scholar 

  28. 28.

    Li J, Culman J, Hortnagl H, Zhao Y, Gerova N, Timm M, Blume A, Zimmermann M et al (2005) Angiotensin AT2 receptor protects against cerebral ischemia-induced neuronal injury. FASEB J 19:617–619

    CAS  Article  Google Scholar 

  29. 29.

    Li JJ, Lu J, Kaur C, Sivakumar V, Wu CY, Ling EA (2009) Expression of angiotensin II and its receptors in the normal and hypoxic amoeboid microglial cells and murine BV-2 cells. Neuroscience 158:1488–1499

    CAS  Article  Google Scholar 

  30. 30.

    Cooney SJ, Bermudez-Sabogal SL, Byrnes KR (2013) Cellular and temporal expression of NADPH oxidase (NOX) isotypes after brain injury. J Neuroinflammation 10:155

    Article  Google Scholar 

  31. 31.

    Choi J, Ifuku M, Noda M, Guilarte TR (2011) Translocator protein (18 kDa)/peripheral benzodiazepine receptor specific ligands induce microglia functions consistent with an activated state. Glia 59:219–230

    Article  Google Scholar 

  32. 32.

    Ye J, Jiang Z, Chen X, Liu M, Li J, Liu N (2015) Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production. Exp Cell Res 340:315–326

    Article  Google Scholar 

  33. 33.

    Xie N, Li H, Wei D, LeSage G, Chen L, Wang S, Zhang Y, Chi L et al (2010) Glycogen synthase kinase-3 and p38 MAPK are required for opioid-induced microglia apoptosis. Neuropharmacology 59:444–451

    CAS  Article  Google Scholar 

  34. 34.

    Wen J, Ribeiro R, Zhang Y (2011) Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells. J Neuroinflammation 8:38. https://doi.org/10.1186/1742-2094-8-38

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Liu B, Hong JS (2003) Role of microglia in inflammation-mediated neurodegenerative diseases: mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther 304:1–7

    CAS  Article  Google Scholar 

  36. 36.

    Bhat SA, Goel R, Shukla R, Hanif K (2017) Platelet CD40L induces activation of astrocytes and microglia in hypertension. Brain Behav Immun 59:173–189. https://doi.org/10.1016/j.bbi.2016.09.021

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Maurya CK, Arha D, Rai AK, Kumar SK, Pandey J, Avisetti DR, Kalivendi SV, Klip A et al (2015) NOD2 activation induces oxidative stress contributing to mitochondrial dysfunction and insulin resistance in skeletal muscle cells. Free Radic Biol Med 89:158–169

    CAS  Article  Google Scholar 

  38. 38.

    Khanna V, Jain M, Singh V, Kanshana JS, Prakash P, Barthwal MK, Murthy PS, Dikshit M (2013) Cholesterol diet withdrawal leads to an initial plaque instability and subsequent regression of accelerated iliac artery atherosclerosis in rabbits. PLoS One 8:e77037

    CAS  Article  Google Scholar 

  39. 39.

    Maehama T, Taylor GS, Slama JT, Dixon JE (2000) A sensitive assay for phosphoinositide phosphatases. Anal Biochem 279:248–250

    CAS  Article  Google Scholar 

  40. 40.

    Lee JW, Lee YK, Yuk DY, Choi DY, Ban SB, Oh KW, Hong JT (2008) Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation. J Neuroinflammation 5:37

    Article  Google Scholar 

  41. 41.

    Lee S, Brait VH, Arumugam TV, Evans MA, Kim HA, Widdop RE, Jones ES (2012) Neuroprotective effect of an angiotensin receptor type 2 agonist following cerebral ischemia in vitro and in vivo. Exp Transl Stroke Med 4:16. https://doi.org/10.1186/2040-7378-4-16

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Xi J, Lei C, Shen L, Chen Z, Xu L, Zhang J, Yu X (2016) Trans-astaxanthin attenuates lipopolysaccharide-induced neuroinflammation and depressive-like behavior in mice. Brain Res 1649(Pt A):30–37

    Google Scholar 

  43. 43.

    Kinoshita D, Cohn DW, Costa-Pinto FA, de Sa-Rocha LC (2009) Behavioral effects of LPS in adult, middle-aged and aged mice. Physiol Behav 96:328–332

    CAS  Article  Google Scholar 

  44. 44.

    Pitychoutis PM, Nakamura K, Tsonis PA, Papadopoulou-Daifoti Z (2009) Neurochemical and behavioral alterations in an inflammatory model of depression: Sex differences exposed. Neuroscience 159:1216–1232

    CAS  Article  Google Scholar 

  45. 45.

    Rojo AI, Innamorato NG, Martin-Moreno AM, De Ceballos ML, Yamamoto M, Cuadrado A (2010) Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson’s disease. Glia 58:588–598

    Article  Google Scholar 

  46. 46.

    Gandhi S, Abramov AY (2012) Mechanism of oxidative stress in neurodegeneration. Oxidative Med Cell Longev 2012:428010

    Article  Google Scholar 

  47. 47.

    Li J, Wuliji O, Li W, Jiang ZG, Ghanbari HA (2013) Oxidative stress and neurodegenerative disorders. Int J Mol Sci 14:24438–24475

    Article  Google Scholar 

  48. 48.

    Uttara B, Singh AV, Zamboni P, Mahajan RT (2009) Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol 7:65–74

    CAS  Article  Google Scholar 

  49. 49.

    Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010) Mechanisms underlying inflammation in neurodegeneration. Cell 140:918–934

    CAS  Article  Google Scholar 

  50. 50.

    Calabrese V, Guagliano E, Sapienza M, Mancuso C, Butterfield DA, Stella AM (2006) Redox regulation of cellular stress response in neurodegenerative disorders. Ital J Biochem 55:263–282

    CAS  PubMed  Google Scholar 

  51. 51.

    Apostolova N, Blas-Garcia A, Esplugues JV (2011) Mitochondria sentencing about cellular life and death: a matter of oxidative stress. Curr Pharm Des 17:4047–4060

    CAS  Article  Google Scholar 

  52. 52.

    Orihuela R, McPherson CA, Harry GJ (2016) Microglial M1/M2 polarization and metabolic states. Br J Pharmacol 173:649–665

    CAS  Article  Google Scholar 

  53. 53.

    Kirchhefer U, Heinick A, König S, Kristensen T, Müller FU, Seidl MD, Boknik P (2014) Protein phosphatase 2A is regulated by protein kinase Cα (PKCα)-dependent phosphorylation of its targeting subunit B56α at Ser41. J Biol Chem 289(1):163–176

    CAS  Article  Google Scholar 

  54. 54.

    Chia KKM, Liu C-C, Hamilton EJ, Garcia A, Fry NA, Hannam W, Figtree GA, Rasmussen HH (2015) Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition. Am J Physiol Cell Physiol 309(4):C239–C250. https://doi.org/10.1152/ajpcell.00392.2014

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Minghetti L, Levi G (1998) Microglia as effector cells in brain damage and repair: Focus on prostanoids and nitric oxide. Prog Neurobiol 54:99–125

    CAS  Article  Google Scholar 

  56. 56.

    Domercq M, Vazquez-Villoldo N, Matute C (2013) Neurotransmitter signaling in the pathophysiology of microglia. Front Cell Neurosci 7:49–176. https://doi.org/10.1074/jbc.M113.507996

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Chien CH, Lee MJ, Liou HC, Liou HH, Fu WM (2016) Microglia-derived cytokines/chemokines are involved in the enhancement of LPS-induced loss of nigrostriatal dopaminergic neurons in DJ-1 knockout mice. PLoS One 11:e0151569

    Article  Google Scholar 

  58. 58.

    Benicky J, Sanchez-Lemus E, Honda M, Pang T, Orecna M, Wang J, Leng Y, Chuang DM et al (2011) Angiotensin II AT1 receptor blockade ameliorates brain inflammation. Neuropsychopharmacology 36:857–870

    CAS  Article  Google Scholar 

Download references

Acknowledgments

We are extremely thankful to Dr. Kumaravelu Jagavelu (CSIR-CDRI) for providing the genotyped WT and KO mice used in the studies. We are highly thankful to Mr. A. L. Vishwakarma, Mrs. M. Chaturvedi, and Mr. Dhananjay Sharma for help with the flow cytometry and confocal microscopy procedures. We are highly thankful to Mr. Jitender Singh Kanshana and Mr. Anant Jaiswal for help in real-time PCR studies. We also acknowledge THUNDER (BSC0102) and MoES (GAP0118) for the confocal facility. The CSIR-CDRI communication number of this article is 9722.

Funding

The study was supported by a financial grant to Kashif Hanif from Department of Biotechnology (DBT, Grant No. BT/PR4021/MED/30/676/2011) and CSIR Network Project MIND (BSC0115). Award of research fellowships to SAB from the Indian Council of Medical Research (ICMR), New Delhi and AS from NIPER, Rae Bareli, are greatly acknowledged.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Kashif Hanif.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Electronic Supplementary Material

ESM 1

(DOC 28066 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bhat, S.A., Sood, A., Shukla, R. et al. AT2R Activation Prevents Microglia Pro-inflammatory Activation in a NOX-Dependent Manner: Inhibition of PKC Activation and p47phox Phosphorylation by PP2A. Mol Neurobiol 56, 3005–3023 (2019). https://doi.org/10.1007/s12035-018-1272-9

Download citation

Keywords

  • AT2 receptor
  • Reactive oxygen species
  • NADPH oxidase
  • PP2A
  • Microglia
  • Neuroinflammation