Keratinocytes express cytokines and nerve growth factor in response to neuropeptide activation of the ERK1/2 and JNK MAPK transcription pathways
Introduction
Skin is densely innervated by sensory neurons that express and release neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), thereby modulating cellular proliferation, wound healing, and pigmentation in the skin [1], [2]. Substance P and CGRP signaling can also stimulate keratinocytes to produce inflammatory mediators such as interleukin-1β (IL-1β) and nerve growth factor (NGF) [3], [4], [5], and it has been proposed that up-regulated keratinocyte expression of inflammatory mediators such as IL-1β, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and NGF contributes to the development of psoriasis, atopic dermatitis, contact dermatitis, post-incisional pain, and complex regional pain syndrome (CRPS) [2], [6], [7], [8], [9]. Furthermore, these conditions are associated with the proliferation of cutaneous neuropeptide containing sensory neurons and neuropeptide receptors, as well as exaggerated neurogenic inflammatory signaling in the affected skin, data suggesting that dysregulated neurocutaneous signaling could cause over-stimulation of the keratinocyte innate immune responses mediating these disease processes [2], [10], [11].
While the ability of neuropeptides to stimulate keratinocyte production of inflammatory mediators has been demonstrated, there is no information concerning the mechanisms by which neuropeptide activation of keratinocyte cell surface receptors ultimately leads to the up-regulation of mediator production. In other cell types SP can induce the phosphorylation of members of the mitogen activated protein kinase (MAPK) family (the extracellular signal related kinase (ERK1/2) and p38) and activate NF-κB. These MAPKs are activated by extracellular stimuli triggering a phosphorylation cascade resulting in nuclear transcription factor activation and inflammatory protein expression. Specific inhibitors of the MAPK signaling pathways have been reported to inhibit SP induced NF-κB activation and chemokine production in macrophages [12], inhibit SP induced IL-6 production in astrocytoma cells [13] and dental pulp cells [14], block SP induced TNF-α expression in mast cells [15], and prevent SP induced TNF-α production in human skin slices [16]. NF-κB is a “rapid-acting” transcription factor that is normally sequestered in an inactive state in the cell cytoplasm, but when stimulated the p50–p65 NF-κB heterodimer is rapidly translocated from the cytoplasm to the nucleus where the p65 NF-κB subunit induces the expression of specific genes involved in inflammation and innate immunity, cell proliferation, response to stress, and apoptosis. Inhibition of NF-κB activation has been reported to block SP induced TNF-α and IL6 production in mast cells [17]. Furthermore, CGRP has been reported to activate MAPK signaling in keratinocytes [18]. Collectively, these results suggest that the MAPK and NF-κB pathways could be involved in the transcriptional regulation of cytokine and NGF over-expression in SP and CGRP stimulated keratinocytes.
In the present study we first attempted to determine whether the neuropeptides SP and CGRP could amplify their own neurocutaneous signal by up-regulating expression of their receptors or even by stimulating keratinocyte expression of these neuropeptides. We also evaluated the stimulatory effects of SP and CGRP signaling on keratinocyte proliferation, and the expression and secretion of proinflammatory cytokines and NGF. We went on to evaluate the roles of the MAPK and NF-κB signaling pathways in supporting neuropeptide stimulated inflammatory mediator responses.
Section snippets
Cell culture
The rat epidermal keratinocyte cell line [19], [20], [21], [22], [23] was generously provided by Dr. Howard Baden (Massachusetts General Hospital, Boston, MA) and cultured as we have previously described [5]. In brief, cells were plated at 1 × 104 cells per 60 mm dish and were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen), supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, 0.4 μg/mL hydrocortisone, and 0.75 mM aminoguanidine. On reaching approximately 80%
Exaggerated neuropeptide signaling in keratinocytes exposed to neuropeptides
The stimulatory effects of SP and CGRP on neuropeptide signaling were tested in an epidermal keratinocyte cell line. Fig. 1A illustrates that SP NK1 receptors are expressed on both the cell membrane and to a degree in the cytoplasm. Furthermore, SP treatment stimulated keratinocyte NK1 receptor expression at both the mRNA (TACR1 gene, Fig. 1B) and protein level, as measured by western immunoblot assay (Fig. 1C). Evidence that SP stimulation of NK1 receptor expression was mediated by MAPK ERK1/2
Discussion
Fig. 1, Fig. 2 illustrate that under normal culture conditions keratinocytes synthesized and secreted low levels of the neurotransmitters SP and CGRP and expressed their cognate receptors. Furthermore, in physiologic concentrations these neuropeptides stimulated keratinocyte expression of the SP and CGRP receptors, and dramatically increase keratinocyte secretion of SP and CGRP. Keratinocyte secreted neuropeptides could have autocrine or paracrine stimulatory effects, potentially amplifying
Acknowledgments
This work was supported by the National Institutes of Health grant NS072168, and the Department of Veterans Affairs, Rehabilitation Research and Development Merit grant F7137R.
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These authors contributed equally to this work.