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Abstract
ABSTRACTPurpose: 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
Highlights
Müller cells are the principal supporting cell in retina[1,2] and in response to retinal disease or injury, they provide a source for regenerated neurons.[3,4] The adult mammalian retina has little or no capacity for regeneration while several cold-blooded vertebrates such as teleost fish functionally regenerate their retina following injury, with Müller cells as a source for new neurons.[5]
The initial events after injury include Müller cell dedifferentiation to a retinal progenitor-like cell and proliferation. This process is regulated by several growth factors including fibroblast growth factors, Wnts and heparin-binding epidermal growth factor (HBEGF) that act by stimulating a core of signaling cascades including mitogen-activated protein kinase (MAPK)/extracellular signalactivated kinases 1/2 (ERK1/2) and phosphatidyl inositide-3 kinase (PI3K) signaling.[6,7,8]
P-epidermal growth factor receptor (EGFR) (Y1173) levels were normalized to EGFR levels and we found a similar pattern of P-EGFR (Y-1173) as P-ERK1/2, with peak levels at 60 min that gradually decreased to background levels by 180 min (Figure 6l)
Summary
Müller cells are the principal supporting cell in retina[1,2] and in response to retinal disease or injury, they provide a source for regenerated neurons.[3,4] The adult mammalian retina has little or no capacity for regeneration while several cold-blooded vertebrates such as teleost fish functionally regenerate their retina following injury, with Müller cells as a source for new neurons.[5] The initial events after injury include Müller cell dedifferentiation to a retinal progenitor-like cell and proliferation. Equipped with the capacity for dedifferentiation but not with the regenerative capacity, the injured mammalian retina will neither get support for the injured neurons from differentiated Müller cells nor it will get newly regenerated neurons. It has been hypothesized that neurons in an injured retina will withstand injury better if the Müller cell dedifferentiation is blocked.[2]
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