Elsevier

Regulatory Peptides

Volume 186, 10 September 2013, Pages 92-103
Regulatory Peptides

Keratinocytes express cytokines and nerve growth factor in response to neuropeptide activation of the ERK1/2 and JNK MAPK transcription pathways

https://doi.org/10.1016/j.regpep.2013.08.001Get rights and content

Highlights

  • Neuropeptides stimulate keratinocytes to secrete SP and CGRP.

  • SP and CGRP stimulate keratinocytes to secrete inflammatory cytokines and NGF.

  • SP activates p38, ERK1/2, and JNK MAPKs and NFκB in keratinocytes.

  • CGRP activates p38 and ERK1/2 MAPKs in keratinocytes.

  • ERK1/2 and JNK signaling mediate keratinocyte inflammatory responses.

Abstract

Sensory neurons innervating the skin can release neuropeptides that are believed to modulate cellular proliferation, wound healing, pigmentation, and keratinocyte innate immune responses. 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 this study we used a keratinocyte cell line to identify the presence of substance P (SP) and calcitonin gene-related peptide (CGRP) receptors on keratinocytes and examined the effects of SP and CGRP stimulation on keratinocyte neuropeptide signaling, cell proliferation, and interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and nerve growth factor (NGF) expression. Neuropeptide stimulation caused an up-regulation of neuropeptide receptor expression in keratinocytes and a dramatic increase in keratinocyte secretion of SP and CGRP, suggesting possible autocrine or paracrine stimulatory effects and amplification of neuropeptide signaling. Both SP and CGRP concentration-dependently stimulated cellular proliferation and the expression and secretion of inflammatory cytokines and NGF in keratinocytes. SP also activated all 3 families of mitogen activated protein kinase (MAPK) and nuclear factor κB (NFκB) in keratinocytes, while CGRP only activated p38 and extracellular signal related kinase1/2 (ERK1/2) MAPKs. Neuropeptide stimulated inflammatory mediatory production in keratinocytes was reversed by ERK1/2 and JNK inhibitors. The current study is the first to observe; 1) that CGRP stimulates keratinocyte expression of CGRP and its receptor complex, 2) that SP and CGRP stimulate IL-6 and TNF-α secretion in keratinocytes, 3) that SP activated all three MAPK families and the NFκB transcriptional signaling pathway in keratinocytes, and 4) that SP and CGRP stimulated inflammatory mediator production in keratinocytes is dependent on ERK1/2 and JNK activation. These studies provide evidence suggesting that disruption of ERK1/2 and JNK signaling may potentially be an effective therapy for inflammatory skin diseases and pain syndromes mediated by exaggerated sensory neuron–keratinocyte signaling.

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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