The Ras/Raf-1/MEK1/ERK signaling pathway coupled to integrin expression mediates cholinergic regulation of keratinocyte directional migration.
ABSTRACT The physiologic mechanisms that determine directionality of lateral migration are a subject of intense research. Galvanotropism in a direct current (DC) electric field represents a natural model of cell re-orientation toward the direction of future migration. Keratinocyte migration is regulated through both the nicotinic and muscarinic classes of acetylcholine (ACh) receptors. We sought to identify the signaling pathway mediating the cholinergic regulation of chemotaxis and galvanotropism. The pharmacologic and molecular modifiers of the Ras/Raf-1/MEK1/ERK signaling pathway altered both chemotaxis toward choline and galvanotropism toward the cathode in a similar way, indicating that the same signaling steps were involved. The galvanotropism was abrogated due to inhibition of ACh production by hemicholinium-3 and restored by exogenously added carbachol. The concentration gradients of ACh and choline toward the cathode in a DC field were established by high-performance liquid chromatographic measurements. This suggested that keratinocyte galvanotaxis is, in effect, chemotaxis toward the concentration gradient of ACh, which it creates in a DC field due to its highly positive charge. A time-course immunofluorescence study of the membrane redistribution of ACh receptors in keratinocytes exposed to a DC field revealed rapid relocation to and clustering at the leading edge of alpha7 nicotinic and M(1) muscarinic receptors. Their inactivation with selective antagonists or small interfering RNAs inhibited galvanotropism, which could be prevented by transfecting the cells with constitutively active MEK1. The end-point effect of the cooperative signaling downstream from alpha7 and M(1) through the MEK1/ERK was an up-regulated expression of alpha(2) and alpha(3) integrins, as judged from the results of real-time PCR and quantitative immunoblotting. Thus, alpha7 works together with M(1) to orient a keratinocyte toward direction of its future migration. Both alpha7 and M(1) apparently engage the Ras/Raf/MEK/ERK pathway to up-regulate expression of the "sedentary" integrins required for stabilization of the lamellipodium at the keratinocyte leading edge.
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ABSTRACT: Integrins are a family of ubiquitous cell surface receptors comprising heterodimers of alpha and beta chains that are required for cell adhesion and motility. Integrin-dependent adhesion and signaling is associated with major conformational changes in the ectodomain as it shifts from a low-affinity "bent" to a high-affinity "extended" structure. The ability of a cell to regulate dynamically the affinity or activation state of an integrin, and hence its binding to extracellular matrix or cell adhesion molecules, is assumed to be driven by intracellular signaling events transmitted by protein binding to the cytoplasmic tail. The binding of an integrin to its ligand can then transmit signals back into the cell to regulate the formation of a macromolecular focal adhesion complex that effectively anchors the cytoskeleton to the adhesion site. Many proteins have been reported to associate physically and functionally with integrins, leading to altered signaling events. A particularly intriguing molecular association exists between integrins and transmembrane proteins that gate the movement of charge, especially voltage-gated potassium channels, although the significance of this interaction is not understood. Although ample evidence indicates that the engagement of integrins can promote potassium efflux by both excitable and nonexcitable cells, we speculate the converse, that the activation state of integrins is dynamically regulated by changes in a transmembrane potential. In this way, direct-current electric fields generated at a site of tissue injury can promote the galvanotaxis or directed migration of cells involved in tissue repair and inflammation.TheScientificWorldJOURNAL 02/2008; 8:1280-94. · 1.66 Impact Factor
Article: Autocrine extracellular signal-regulated kinase activation in normal human keratinocytes is not interrupted by calcium triggering and is involved in the control of cell cycle at the early stage of calcium-induced differentiation.[show abstract] [hide abstract]
ABSTRACT: Normal human epidermal keratinocytes (NHEK) respond to the autocrine activated extracellular signal-regulated kinase (ERK) signaling pathway, which contributes to the survival of keratinocytes. However, during the condition of calcium-induced differentiation, how the autocrine ERK signaling is regulated and affected is poorly understood. The purpose of this study was to understand and to obtain clues to the possible function of the autocrine ERK activation during the calcium-induced differentiation of NHEK. We demonstrated that the autocrine activated ERK was not interrupted by calcium triggering and that it was sustained for at least one day after changing the medium. We also found that the autocrine ERK activation was associated with the expression of cyclin D1 and the cell cycle regulation at the early stage of calcium triggering by treating the cells with the mitogen-activated protein kinase inhibitor PD98059. However, the PD98059 treatment did not have a significant influence on the expression of involucrin and loricrin. In addition, we demonstrated that autocrine ERK activation was associated with protein kinase C and p38MAPK signaling. We suggest that the activation of autocrine ERK is not interrupted by calcium triggering and it might participate in cell growth during the early stage of calcium-induced differentiation in NHEK.Journal of Korean Medical Science 05/2007; 22(2):290-7. · 0.99 Impact Factor
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ABSTRACT: This review focuses on the role of the peripheral nervous system in cutaneous biology and disease. During the last few years, a modern concept of an interactive network between cutaneous nerves, the neuroendocrine axis, and the immune system has been established. We learned that neurocutaneous interactions influence a variety of physiological and pathophysiological functions, including cell growth, immunity, inflammation, pruritus, and wound healing. This interaction is mediated by primary afferent as well as autonomic nerves, which release neuromediators and activate specific receptors on many target cells in the skin. A dense network of sensory nerves releases neuropeptides, thereby modulating inflammation, cell growth, and the immune responses in the skin. Neurotrophic factors, in addition to regulating nerve growth, participate in many properties of skin function. The skin expresses a variety of neurohormone receptors coupled to heterotrimeric G proteins that are tightly involved in skin homeostasis and inflammation. This neurohormone-receptor interaction is modulated by endopeptidases, which are able to terminate neuropeptide-induced inflammatory or immune responses. Neuronal proteinase-activated receptors or transient receptor potential ion channels are recently described receptors that may have been important in regulating neurogenic inflammation, pain, and pruritus. Together, a close multidirectional interaction between neuromediators, high-affinity receptors, and regulatory proteases is critically involved to maintain tissue integrity and regulate inflammatory responses in the skin. A deeper understanding of cutaneous neuroimmunoendocrinology may help to develop new strategies for the treatment of several skin diseases.Physiological Reviews 11/2006; 86(4):1309-79. · 26.87 Impact Factor