Search

Many aspects of neuronal development, such as neuronal survival and differentiation, are regulated by the transcription factor cAMP-response element-binding protein (CREB). We have previously reported that α 2 -adrenergic receptors (ARs), members of the G protein-coupled receptor (GPCR) superfamily, induce neuronal differentiation of rat pheochromocytoma (PC)-12 cells in a subtype-specific manner, i.e. α 2A <α 2B <α 2C . Herein, we sought to investigate CREB`s involvement in this α 2 AR-dependent neurogenic process. We used a combination of gene reporter assays and immunoblotting analysis, coupled with co-immunoprecipitation and morphological analysis, in transfected PC12 cell lines. Chronic α 2B - or α 2C -AR activation results in sustained CREB activation, which is both necessary and sufficient for neuronal differentiation of PC12 cells expressing these two α 2 ARs. In contrast, chronic α 2A activation only leads to transient CREB activation, insufficient for PC12 neuronal differentiation. However, upon CREB overexpression, α 2A -AR triggers neuronal differentiation similarly to α 2B - or α 2C -ARs. Importantly, NGF (Nerve Growth Factor)`s TrkA receptor transactivation is essential for the sustained activation of CREB by all three α 2 subtypes in PC12 cells, whereas protein kinase A (PKA), the prototypic kinase that phosphorylates CREB, is not. Instead, TrkA-activated GPCR-kinase (GRK)-2 mediates the sustained CREB activation during α 2 AR-induced neuronal differentiation of PC12 cells. In conclusion, catecholaminergic-induced neuronal differentiation of PC12 cells through α 2 ARs uses a non-canonical pathway involving TrkA transactivation and subsequent GRK2-dependent, sustained phosphorylation/activation of CREB. These findings provide novel insights into catecholaminergic neurogenesis and suggest that α 2 AR agonists, combined with NGF analogs or GRK2 stimulators, may exert neurogenic/neuroprotective effects., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The cytotoxicity of cigarette smoke (CS) in humans is well-documented, but the mechanism behind CS toxicity and carcinogenicity remains unknown. We are interested in the toxicological effects of CS gas phase and the biological mechanisms of its action., Materials and Methods: Gas phase CS cytotoxicity was measured by Wst-1 and LDH assays, in cultured cells. The mechanism of cell death was investigated by flow cytometric analysis using Annexin V and PI staining. Gas phase CS-induced oxidative damage was evaluated by estimating cellular glutathione (GSH) levels. Protein modifications (nitration of tyrosines) induced by gas phase CS and activation of key signalling proteins (Mitogen-activated protein kinase, MAPK) were detected by immunoblotting., Results: The cytotoxicity of gas phase CS was found to be dose-dependent. The mechanism of cell death was found to be both apoptotic and necrotic depending on the concentrations used. Exposure to gas phase CS resulted in depletion of cellular GSH levels, increased nitrotyrosine immunoreactivity and phosphorylation of p44/42 MAPK proteins., Conclusion: These results suggest that the CS gas phase alone contributes significantly to the deleterious effects of CS in cellular systems.