Beta-adrenergic receptor activation inhibits keratinocyte migration via a cyclic adenosine monophosphate-independent mechanism.
ABSTRACT There is increasing evidence that G-protein-coupled receptors cross-talk with growth factor receptor-mediated signal transduction in a variety of cell types. We have investigated mechanisms by which the activation of beta-adrenergic receptors, classically GTP-binding proteins coupled receptors, influence the migration of cultured human keratinocytes. We found that iso-proterenol, a beta-adrenergic receptor-selective agonist, inhibited cell migration stimulated by either epidermal growth factor, or extracellular Ca2+ in a concentration-dependent manner. This was prevented by pretreatment of the cells with the beta-adrenergic receptor-selective antagonist timolol. Interestingly, isoproterenol, at a concentration of 1 nm, did not measurably increase intracellular cyclic adenosine monophosphate concentrations yet inhibited cell migration by 50%. To test further if isoproterenol's actions were mediated via activation of adenylyl cyclase, two inhibitors of its activity, 2'5'-dideoxyadenosine and SQ22536, were used. Both compounds significantly diminished iso-proterenol-induced increases in intracellular cyclic adenosine monophosphate concentrations but did not attenuate isoproterenol-induced inhibition of cell migration. Also, forskolin (1 microm) markedly increased intracellular cyclic adenosine monophosphate concentrations but did not significantly inhibit cell migration. As mitogen-activated protein kinases are known to signal growth factor-stimulated cell migration, we examined whether beta-adrenergic receptor-mediated inhibition of keratinocyte migration might occur via inactivation of mitogen-activated protein kinases. We found that isoproterenol inhibited phosphorylation of extracellular signal-regulated kinase mitogen-activated protein kinase in a concentration-dependent manner but had no effect on the phosphorylation of the stress mitogen-activated protein kinases c-jun N-terminal kinase and stress-activated protein kinase-2. Neither forskolin nor a membrane permeable cyclic adenosine monophosphate analog inhibited phosphorylation of any of these mitogen-activated protein kinases. These findings suggest that beta-adrenergic receptor-induced inhibition of keratinocyte migration is mediated through inhibition of the extracellular signal-regulated kinase mitogen-activated protein kinase signaling in a cyclic adenosine monophosphate-independent manner.
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ABSTRACT: The EGFR-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that the absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are markedly reduced, and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this previously unreported function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the ESCRT (endosomal sorting complex required for transport) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors.Journal of Investigative Dermatology advance online publication, 12 July 2012; doi:10.1038/jid.2012.211.Journal of Investigative Dermatology 07/2012; · 6.19 Impact Factor
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ABSTRACT: β-Adrenergic receptor (βAR) agonists reduce intraocular pressure (IOP) by increasing aqueous outflow through the trabecular meshwork (TM). However, although this effect is well established, the specific signaling mechanisms responsible are less clear. To address this, the current study examined βAR signaling in primary human trabecular meshwork cells (HTMCs), specifically, focusing on the effect of βAR activation on the extracellular signal regulated kinases 1/2 (ERK). HTMCs were cultured and assessed for βAR expression by both immunofluorescence and reverse-transcription-polymerase chain reaction. The effect of βAR activation on ERK phosphorylation was examined in these cells by In-Cell Western™ analysis. Pharmacological approaches were used to characterize the mechanism of the βAR effect on ERK. Treatment of HTMCs with the nonselective βAR agonist, isoproterenol (ISO), decreased the basal phospho-ERK (pERK) level, through actions at the β2AR. The response was insensitive to pertussis toxin (PTx), but pretreatment with cholera toxin (CTx) resulted in a reversal of the response, such that ISO treatment instead increased pERK, thus implicating Gα(s) in the inhibitory pERK response. The adenylyl cyclase activator, forskolin, also decreased pERK, suggesting the involvement of adenylyl cyclase and cAMP, whereas a protein kinase A (PKA) inhibitor, H-89, blocked both ISO and forskolin-mediated pERK inhibition in HTMCs. Finally, a closer examination of the pERK increase generated in the presence of CTx demonstrated that it was also insensitive to PTx, and appeared to have differing rank orders of efficacy for various βAR agonists compared with the inhibitory pERK pathway in HTMCs. A novel β(2)AR-signaling pathway leading to a decrease in pERK, which was dependent on Gα(s), cAMP, and PKA, was identified in HTMCs. A competing β(2)AR signaling pathway resulting in increased pERK was also identified. Since βAR effects on aqueous humor (AH) outflow have been linked to cAMP, and inhibition of ERK in TM cells has recently been suggested as increasing AH outflow, our findings suggest that the inhibitory β(2)AR-pERK pathway likely represents the mechanism by which βAR agonists decrease IOP. The presence of a competing β(2)AR-ERK activation pathway in the same cells suggests that this is an ideal system for the development and validation of functionally selective β(2)AR agonists.Journal of ocular pharmacology and therapeutics: the official journal of the Association for Ocular Pharmacology and Therapeutics 02/2012; 28(1):17-25. · 1.46 Impact Factor
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ABSTRACT: Lichen planus-like drug eruptions (LDE) can appear similar or identical to idiopathic lichen planus. We present a 45-year-old man with a widespread, violaceous, papular, generalized exanthema with histologic features of a lichenoid reaction, which subsequently resolved with the cessation of labetatol. We found 29 cases of previously reported β-adrenoceptor antagonist (β-blocker)-associated LDE. This is a relatively rare complication that may present as classic lichenoid papules indistinguishable from lichen planus and has a predilection for the limbs, chest, back, and oral mucosa. Histologically, there is a lichenoid infiltrate often with eosinophils. LDE may be due to drug cross-reactivity or as a result of a suppressed skin adrenergic system. Multiple potential medications in case studies and the inability to differentiate LDE from idiopathic lichen planus in cross-sectional association studies make any conclusive analysis difficult.American Journal of Clinical Dermatology 07/2012; 13(6):417-21. · 2.52 Impact Factor