Cordula Kempkes

A sensory neuron–expressed IL-31 receptor mediates T helper cell–dependent itch: Involvement of TRPV1 and TRPA1

BACKGROUND Although the cytokine IL-31 has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear. OBJECTIVE We sought to determine whether immune cell-derived IL-31 directly stimulates sensory neurons and to identify the molecular basis of IL-31-induced itch. METHODS We used immunohistochemistry and quantitative real-time PCR to determine IL-31 expression levels in mice and human subjects. Immunohistochemistry, immunofluorescence, quantitative real-time PCR, in vivo pharmacology, Western blotting, single-cell calcium imaging, and electrophysiology were used to examine the distribution, functionality, and cellular basis of the neuronal IL-31 receptor α in mice and human subjects. RESULTS Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and, to a significantly lesser extent, mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentrations increased significantly in murine atopy-like dermatitis skin. Both human and mouse dorsal root ganglia neurons express IL-31RA, largely in neurons that coexpress transient receptor potential cation channel vanilloid subtype 1 (TRPV1). IL-31-induced itch was significantly reduced in TRPV1-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)-deficient mice but not in c-kit or proteinase-activated receptor 2 mice. In cultured primary sensory neurons IL-31 triggered Ca(2+) release and extracellular signal-regulated kinase 1/2 phosphorylation, inhibition of which blocked IL-31 signaling in vitro and reduced IL-31-induced scratching in vivo. CONCLUSION IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA(+)/TRPV1(+)/TRPA1(+) neurons and is a critical neuroimmune link between TH2 cells and sensory nerves for the generation of T cell-mediated itch. Thus targeting neuronal IL-31RA might be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T-cell lymphoma.

UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling

Significance Skin protects against harmful external cues, one of them UV radiation, which, upon overexposure, causes sunburn as part of the UVB response. Using genetically engineered mice and cultured skin epithelial cells, we have identified the calcium-permeable TRPV4 ion channel in skin epithelial cells as critical for translating the UVB stimulus into intracellular signals and also into signals from epithelial skin cell to sensory nerve cell that innervates the skin, causing pain. These signaling mechanisms underlie sunburn and in particular sunburn-associated pain. Thus, activation of TRPV4 in skin by UVB evokes sunburn pain, highlighting the forefront-signaling role of the skin and TRPV4. At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca2+ response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.

Involvement of TRPV4 in Serotonin-Evoked Scratching.

Several thermo-sensitive TRP channels (TRPV1, -3; TRPA1) have been implicated in itch. In contrast, the role of transient receptor potential vanilloid type-4 (TRPV4) in itch is unknown. Therefore, we investigated if TRPV4, a temperature-sensitive cation channel, plays an important role in acute itch in mice. Four different pruritogens including serotonin (5-hydroxytrytamine, 5-HT), histamine, SLIGRL (PAR2/MrgprC11 agonist) and chloroquine (MrgprA3 agonist) were intradermally injected and itch-related scratching behavior was assessed. TRPV4 knockout (TRPV4KO) mice exhibited significantly fewer 5-HT-evoked scratching bouts compared to wild-type (WT) mice. Notably, no differences between TRPV4KO and WT mice were observed in the number of scratch bouts elicited by SLIGRL and histamine. Pretreatment with a TRPV4 antagonist significantly attenuated 5-HT-evoked scratching in vivo. Using calcium imaging in cultured primary murine dorsal root ganglion (DRG) neurons, the response of neurons after 5-HT application, but not other pruritogens, was significantly lower in TRPV4KO compared to WT mice. A TRPV4 antagonist significantly suppressed 5-HT-evoked responses in DRG cells from WT mice. Approximately 90% of 5-HT-sensitive DRG neurons were immunoreactive for an antibody to TRPV4, as assessed by calcium imaging. These results indicate that serotonin-induced itch is linked to TRPV4.

Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

Neural peptidase endothelin-converting enzyme 1 regulates endothelin 1–induced pruritus

In humans, pruritus (itch) is a common but poorly understood symptom in numerous skin and systemic diseases. Endothelin 1 (ET-1) evokes histamine-independent pruritus in mammals through activation of its cognate G protein-coupled receptor endothelin A receptor (ETAR). Here, we have identified neural endothelin-converting enzyme 1 (ECE-1) as a key regulator of ET-1-induced pruritus and neural signaling of itch. We show here that ETAR, ET-1, and ECE-1 are expressed and colocalize in murine dorsal root ganglia (DRG) neurons and human skin nerves. In murine DRG neurons, ET-1 induced internalization of ETAR within ECE-1-containing endosomes. ECE-1 inhibition slowed ETAR recycling yet prolonged ET-1-induced activation of ERK1/2, but not p38. In a murine itch model, ET-1-induced scratching behavior was substantially augmented by pharmacological ECE-1 inhibition and abrogated by treatment with an ERK1/2 inhibitor. Using iontophoresis, we demonstrated that ET-1 is a potent, partially histamine-independent pruritogen in humans. Immunohistochemical evaluation of skin from prurigo nodularis patients confirmed an upregulation of the ET-1/ETAR/ECE-1/ERK1/2 axis in patients with chronic itch. Together, our data identify the neural peptidase ECE-1 as a negative regulator of itch on sensory nerves by directly regulating ET-1-induced pruritus in humans and mice. Furthermore, these results implicate the ET-1/ECE-1/ERK1/2 pathway as a therapeutic target to treat pruritus in humans.

Role of Matriptase and Proteinase-Activated Receptor-2 in Nonmelanoma Skin Cancer

Matriptase (membrane-type serine proteinase) was reported to play a role in nonmelanoma skin cancer progression. Moreover, it was shown to stimulate proteinase-activated receptor-2 (PAR(2)) in vitro. Hepatocyte growth factor activator inhibitor-1 (HAI-1), the matriptase inhibitor, is an important regulator of enzyme activity. Therefore, the aim of this study was to elucidate the putative role of matriptase, HAI-1, and PAR(2) in normal human skin, as well as in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). In normal human epidermis, PAR(2) colocalized with matriptase and HAI-1. Immunoreactivity of all proteins was found to be diminished in BCCs. Likewise, PAR(2) immunoreactivity was significantly decreased, whereas matriptase immunoreactivity was enhanced with SCC progression. We could also show that matriptase was complexed to HAI-1 in normal human skin, whereas in SCCs, the enzyme was present in an unassociated form. Both a specific peptide agonist for PAR(2) and the proteinase domain of matriptase were able to induce intracellular calcium mobilization and inhibition of proliferation in cultured HaCaT keratinocytes. In conclusion, our results suggest that PAR(2) is a substrate for matriptase in human skin in vivo. Deregulation of these proteins delineates SCC progression.

Role of protease-activated receptors in human skin fibrosis and scleroderma

Abstract:  Proteases and their receptors have poorly understood roles in skin fibrosis and systemic scleroderma (SSc). We examined the role of protease‐activated receptors (PAR1 and PAR2) in the pathophysiology of human SSc and skin fibrosis. Immunohistochemistry showed that PAR1 immunoreactivity was positive in fibroblasts of SSc skin and healthy skin. PAR2 immunoreactivity was positive in SSc skin, but negative in endothelial cells and fibroblasts of healthy skin. Double immunofluorescence using an antibody against smooth muscle actin (α‐SMA) as a marker for myofibroblasts verified a certain percentage of myofibroblasts positive for PAR1 and PAR2 in SSc skin. In human dermal cultured fibroblasts (HDF), PAR1 stimulation with or without bleomycin pretreatment mobilized intracellular calcium, indicating that the expressed PARs are functional and have effects on downstream signalling by calcium release. PAR2‐induced intracellular calcium mobilization was only measurable in HDF after bleomycin pretreatment. Thus, PAR1‐ and PAR2‐positive fibroblasts are increased in SSc, indicating a regulatory role. Intriguingly, bleomycin activated PAR2 in HDF indicating that fibrosis‐promoting factors have a direct effect on PAR2 expression and functionality.

Proteinase-Activated Receptors 1 and 2 Regulate Invasive Behavior of Human Melanoma Cells via Activation of Protein Kinase D1

Recent studies have indicated an important role of proteinases and proteinase-activated receptors (PARs) in tumorigenesis. Although a role for PARs has been described in various skin tumors including melanoma, the underlying cellular mechanisms have not been understood. Recent studies have suggested PAR(1) as a regulator of melanoma cell growth and metastasis by affecting angiogenic and invasive factors. Moreover, changes in the expression patterns of PAR(1) and PAR(2) correlate with skin cancer progression, and PAR(1) is overexpressed in melanoma. Therefore, we sought to elucidate the putative role of PAR(1)- and PAR(2)-mediated signal transduction pathways during melanoma progression. Activation of both PAR(1) and PAR(2) led to rapid phosphorylation of protein kinase D1 (PKD1) in cultured WM9 melanoma cells. PKD1 is known to be involved in cell migration, integrin regulation, and intracellular vesicle transport. Downregulation of PKD1 by siRNA resulted in diminished proliferation, decreased αvβ3 integrin regulation, and secretion of pro-angiogenic chemokine IL-8 in WM9 cells. In conclusion, our results show that PAR(1) and PAR(2) are involved in WM9 cell proliferation and secretion of IL-8 by activation of PKD1. Inactivation of the PKD1 pathway may be beneficial for the inhibition of PAR-induced melanoma proliferation and for maintenance of the inflammatory tumor environment.