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Alpha 2-Adrenergic Receptor Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling via Transactivation of EGF Receptors in the Human MIO-M1 Müller Cell Line
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Northeast Ohio Med Univ, Dept Pharmaceut Sci, Rootstown, OH USA. (Finn Hallböök group)ORCID iD: 0000-0003-1549-2418
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.ORCID iD: 0000-0001-7552-187X
2019 (English)In: Current Eye Research, ISSN 0271-3683, E-ISSN 1460-2202, Vol. 44, no 1, p. 34-45Article in journal (Refereed) Published
Abstract [en]

Purpose: Alpha 2-adrenergic receptor (α2-ADR) agonists are used clinically for a range of indications including reducing elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Animal experiments show that α2-ADR agonists attenuate the injury-induced Müller cell dedifferentiation by a mechanism that involves activation and regulation of extracellular signal-regulated kinase (ERK) 1/2 leading to transactivation of epidermal growth factor receptors (EGFRs). The purpose of this study was to study and corroborate the activation of this system in human cells.

Material and Methods: The human Müller cell line MIO-M1 was treated with the α2A-ADR agonist brimonidine in combination with inhibitors for Src-kinase, EGFR-kinase, matrix metalloproteinase (MMP) as well as small interfering RNAs (siRNAs) for the EGFR. The cells were analyzed using immunocytochemistry, quantitative PCR and western blot techniques.

Results: Our results show that human MIO-M1 cells express α2A-ADRs and that stimulation of these receptors caused a robust increase of ERK1/2 and protein kinase B (PKB/AKT) (Thr-308) phosphorylation in MIO-M1 cells. P-ERK1/2 and P-AKT (Thr-308) signaling was mediated by Src-kinase and associated with phosphorylation of tyrosine residue of epidermal growth factor receptor (P-EGFR Y1173). In addition, the agonist caused activation of MMPs. These effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-kinase inhibitor (AG1478), EGFR-siRNA and a MMP inhibitor (GM6001).

Conclusion: The results confirm that this human Müller cell line responds to ADR stimulation with phosphorylation of ERK and AKT, which suggests that it is possible to pharmacologically target ADR to modulate the early events in human Müller cell dedifferentiation in a similar fashion as been shown for chicken Müller cells.

Abbreviations: CRALBP: cellular retinaldehyde binding protein; EGFR: epidermal growth factor receptor; ERK1/2: extracellular signal-regulated kinase 1/2; GS: glutamine synthetase; GPCR: G protein-coupled receptor; IR: immunoreactivity; MAPK: mitogen-activated protein kinase; MMP: matrix metalloproteinase; P-ERK1/2: phospho-ERK1/2; qRT-PCR: quantitative reverse transcriptase PCR

Place, publisher, year, edition, pages
2019. Vol. 44, no 1, p. 34-45
Keywords [en]
AKT pathway, Alpha 2-adrenergic receptors, Brimonidine, EGF receptor, ERK1/2, Matrix metalloproteinases, MIO-M1 human Müller cell, and Src-kinase
National Category
Neurosciences
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-281576DOI: 10.1080/02713683.2018.1516783ISI: 000454952100006PubMedID: 30198788OAI: oai:DiVA.org:uu-281576DiVA, id: diva2:914631
Funder
Swedish Research Council, 2016-01641
Note

Title in thesis list of papers: Alpha2-Adrenergic Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling Via Transactivation of EGF Receptors in MIO-M1 Human Müller Cells

Available from: 2016-03-24 Created: 2016-03-24 Last updated: 2019-01-25Bibliographically approved
In thesis
1. Modulation of the Progenitor Cell and Homeostatic Capacities of Müller Glia Cells in Retina: Focus on α2-Adrenergic and Endothelin Receptor Signaling Systems
Open this publication in new window or tab >>Modulation of the Progenitor Cell and Homeostatic Capacities of Müller Glia Cells in Retina: Focus on α2-Adrenergic and Endothelin Receptor Signaling Systems
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Müller cells are major glial cells in the retina and have a broad range of functions that are vital for the retinal neurons. During retinal injury gliotic response either leads to Müller cell dedifferentiation and formation of a retinal progenitor or to maintenance of mature Müller cell functions. The overall aim of this thesis was to investigate the intra- and extracellular signaling of Müller cells, to understand how Müller cells communicate during an injury and how their properties can be regulated after injury. Focus has been on the α2-adrenergic receptor (α2-ADR) and endothelin receptor (EDNR)-induced modulation of Müller cell-properties after injury.

The results show that α2-ADR stimulation by brimonidine (BMD) triggers Src-kinase mediated ligand-dependent and ligand-independent transactivation of epidermal growth factor receptor (EGFR) in both chicken and human Müller cells. The effects of this transactivation in injured retina attenuate injury-induced activation and dedifferentiation of Müller cells by attenuating injury-induced ERK signaling. The attenuation was concomitant with a synergistic up-regulation of negative ERK- and RTK-feedback regulators during injury. The data suggest that adrenergic stress-signals modulate glial responses during retinal injury and that α2-ADR pharmacology can be used to modulate glial injury-response. We studied the effects of this attenuation of Müller cell dedifferentiation on injured retina from the perspective of neuroprotection. We analyzed retinal ganglion cell (RGC) survival after α2-ADR stimulation of excitotoxically injured chicken retina and our results show that α2-ADR stimulation protects RGCs against the excitotoxic injury. We propose that α2-ADR-induced protection of RGCs in injured retina is due to enhancing the attenuation of the glial injury response and to sustaining mature glial functions. Moreover, we studied endothelin-induced intracellular signaling in Müller cells and our results show that stimulation of EDNRB transactivates EGFR in Müller cells in a similar way as seen after α2-ADR stimulation. These results outline a mechanism of how injury-induced endothelins may modulate the gliotic responses of Müller cells.

The results obtained in this thesis are pivotal and provide new insights into glial functions, thereby uncovering possibilities to target Müller cells by designing neuroprotective treatments of retinal degenerative diseases or acute retinal injury.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. p. 73
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1201
Keywords
Alpha2-adrenergic receptor, Brimonidine, Brn3a, Dedifferentiation, Endothelin, EGFR, ERK1/2, Neuroprotection, NMDA, MIO-M1 human Müller cell, Müller cells, Retina, Retinal ganglion cells, Src-kinase, Transactivation.
National Category
Neurosciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-281569 (URN)978-91-554-9527-5 (ISBN)
Public defence
2016-05-19, B21, BMC, Husagatan 03, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-04-28 Created: 2016-03-24 Last updated: 2018-01-10

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Harun-Or-Rashid, MohammadHallböök, Finn

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