Cyclic AMP mediates keratinocyte directional migration in an electric field

Abstract

Re-epithelialization of wounded skin is necessary for wound closure and restoration of barrier function and requires directional keratinocyte migration towards the center of the wound. The electric field (EF) generated immediately upon wounding could be the earliest signal keratinocytes receive to initiate directional migration and healing. Keratinocytes express many β2-adrenergic receptors (β2-ARs), but their role in the epidermis is unknown. We have previously shown that β-AR agonists decrease keratinocyte migration in a cyclic AMP (cAMP) independent mechanism involving the activation of protein phosphatase 2A (PP2A). Here, we ask whether β2-ARs play a role in keratinocyte galvanotaxis. We report a bimodal response. When keratinocytes were exposed to higher concentrations of β-AR agonist (0.1 μM), their tracked migratory speed was inhibited, in both the presence (directional migration) and the absence (random migration) of a 100 mV mm–1 EF, as expected. At lower agonist concentrations (0.1 pM to 0.1 nM), there was no effect on migratory speed; however, all directionality was lost – essentially, cells were `blinded' to the directional cue. Preincubating the cells with β-antagonist restored directional migration, demonstrating that the `blindness' was β2-AR mediated. Incubation of keratinocytes with agents known to increase intracellular cAMP levels, such as sp-cAMP, pertussis toxin and forskolin, resulted in similar `blinding' to the EF, whereas random migration was unaffected. The inactive cAMP analog rp-cAMP had no effect on keratinocyte migration, whether directional or random. However, rp-cAMP pretreatment before β-agonist addition fully restored galvanotaxis, demonstrating the complete cAMP dependence of the attenuation of keratinocyte directional migration. This is the first report that cAMP is capable of mediating keratinocyte galvanotaxis. β-AR agonists and antagonists could be valuable tools for modulating re-epithelialization, an essential step in the wound-healing process. Thus, β-ARs regulate the two distinct components of keratinocyte directional migration differently: migration speed via a cAMP-independent mechanism and galvanotaxis by a cAMP-dependent one.

The β2-adrenergic receptor activates pro-migratory and pro-proliferative pathways in dermal fibroblasts via divergent mechanisms

“…We discovered that in contrast to the anti-motogenic effects of ␤2-AR activation in keratinocytes (Pullar et al, 2003;Pullar and Isseroff, 2005), the activation of ␤2-AR in dermal fibroblasts was both pro-motogenic and pro-mitogenic. The diametrically opposite response to ␤2-AR activation in fibroblasts as compared to keratinocytes underscores the importance of evaluating the ␤2-AR-mediated responses in a specific cell type.…”

Electrophoresis of cell membrane heparan sulfate regulates galvanotaxis in glial cells

“…We hypothesize that one of the contributing factors in mediating the competing mechanisms may be the cellular level of cAMP, as poly-L-lysine was shown to elevate cellular cAMP levels more than twofold in oligodendrocytes (Vartanian et al, 1988). Cellular levels of cAMP have been shown to modulate the repulsive versus attractive response towards netrin-1 in Xenopus spinal neurons (Ming et al, 1997) and elevated cAMP levels abolished the galvanotaxis of keratinocytes and keratocyte fragments (Pullar and Isseroff, 2005;Zhu et al, 2015). Another candidate in regulating the anodic galvanotaxis is sialic acid, a negatively charged sugar molecule that contributes to the overall charge of the membrane.…”

Beta Adrenergic Receptors in Keratinocytes

“…This is in contradistinction to beta2AR activation in multiple other cell types, which typically results in ERK phosphorylation and activation [34][35][36][37]. Classical, cAMP-dependent signaling with beta2AR activation has also been described in keratinocytes [38,39]. Thus, both cAMP -dependent and independent signal transduction govern keratinocyte physiology, and continuing investigations will clarify which pathway controls which cellular functions.…”