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Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons.

ABSTRACT: BACKGROUND AND PURPOSE: Stimulation of astrocytes by the alpha(2)-adrenoceptor agonist dexmedetomidine, a neuroprotective drug, transactivates epidermal growth factor (EGF) receptors. The present study investigates signal pathways leading to release of an EGF receptor ligand and those activated during EGF receptor stimulation, and the response of neurons to dexmedetomidine and to astrocyte-conditioned medium. EXPERIMENTAL APPROACH: Phosphorylation of ERK(1/2) was determined by western blotting and immunocytochemistry, and phosphorylation of EGF receptors by immunoprecipitation and western blotting. mRNA expression of fos family was measured by RT-PCR. KEY RESULTS: Pertussis toxin (0.2 microg ml(-1)) an inhibitor of betagamma subunit dissociation from Galpha(i) protein, and GF 109203X (500 nM), a protein kinase C inhibitor, abolished ERK(1/2) phosphorylation. PP1 (10 microM), inhibiting Src kinase and GM 6001 (10 microM), an inhibitor of Zn-dependent metalloproteinase, abolished ERK(1/2) phosphorylation by dexmedetomidine (50 nM), but not that by EGF (10 ng ml(-1)), showing Src kinase and metalloproteinase activation during the first stage only; AG 1478 (1 microM), an inhibitor of the EGF receptor tyrosine kinase, abolished ERK(1/2) phosphorylation. Dexmedetomidine-induced EGF receptor phosphorylation was prevented by AG 1478, GM 6001, PP1 and GF 109203X and its induction of cfos and fosB by AG 1478 and by U0126 (10 microM), an inhibitor of ERK phosphorylation, indicating downstream effects of ERK(1/2) phosphorylation. EGF and conditioned medium from dexmedetomidine-treated astrocytes, but not dexmedetomidine itself, induced ERK phosphorylation in primary cultures of cerebellar neurons. CONCLUSIONS AND IMPLICATIONS: Dexmedetomidine-induced transactivation pathways were delineated. Its paracrine effect on neurons may account for its neuroprotective effects.

SUBMITTER: Li B

PROVIDER: S-EPMC2438992 | biostudies-other | 2008 May

REPOSITORIES: biostudies-other

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Project description:Injury to the eye or retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the capacity to generate new neurons. The epidermal growth factor receptor (EGFR) system and extracellular signal-regulated kinase (ERK) signaling are key regulators of these processes in Müller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whether endothelin receptor signaling can activate EGFR and ERK signaling in Müller cells. Endothelin expression is robustly upregulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell types. We analyzed the endothelin signaling system in chicken retina and cultured primary chicken Müller cells as well as the human Müller cell line MIO-M1. The Müller cells were stimulated with receptor agonists and treated with specific blockers to key enzymes in the signaling pathway or with siRNAs. We focused on endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that chicken Müller cells and the human Müller cell line MIO-M1 express endothelin receptor B. Stimulation by the endothelin receptor B agonist IRL1620 triggered phosphorylation of ERK1/2 and autophosphorylation of (Y1173) EGFR. The effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-inhibitor (AG1478), EGFR-siRNA and by inhibitors to extracellular matrix metalloproteinases (GM6001), consistent with a Src-kinase mediated endothelin receptor response that engage ligand-dependent and ligand-independent EGFR activation. Our data suggest a mechanism for how injury-induced endothelins, produced in the retina, may modulate the Müller cell responses by Src-mediated transactivation of EGFRs. The data give support to a view in which endothelins among several other functions, serve as an injury-signal that regulate the gliotic response of Müller cells.

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Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons.

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