Endothelin-1 Participates in the Pathogenesis of Prurigo Nodularis by Promoting NGF Expression via Endothelial Receptor B in Epidermal Keratinocytes and Dorsal Root Ganglion Cells

Abstract

Prurigo nodularis (PN) is a debilitating chronic skin disorder characterized by intractable itch and nodular lesions [1]. While its exact etiology remains unclear, emerging evidence suggests a complex interplay between neuronal sensitization and cutaneous changes [2]. Recent studies indicate increased epidermal branching and heightened expression of neuroplasticity-related genes regulating sensory neuron growth in PN [3]. Notably, nerve growth factor (NGF), a neurotrophic factor essential for nociceptive signaling [4], is highly expressed in PN compared to atopic dermatitis (AD) and brachioradial pruritus. Its elevated expression correlates with increased epidermal nerve fiber branching [3]. This NGF-mediated neuroplasticity may contribute to neuronal alterations in PN.

In our previous study, we identified significantly elevated endothelin-1 (EDN1) expression in PN lesional skin and serum, suggesting its role in local and systemic modulation [5]. Recent single-cell analysis further revealed increased EDN1 expression in PN compared to AD and healthy controls, underscoring its disease-specific significance [6]. While EDN1 is recognized as a histamine-independent pruritogen [7], its role in neural sensitization remains unclear. In particular, its direct impact on dorsal root ganglion (DRG) neurons has not been explored. Here, we investigate the interaction between EDN1 and NGF in keratinocytes and DRG neurons, contributing to neuronal sensitization in PN.

To elucidate the role of NGF, we assessed NGF and brain-derived neurotrophic factor (BDNF) expression in PN lesional skin, as BDNF, another neurotrophic factor involved in neuronal growth and sensitization, was assessed for comparison. Immunofluorescence revealed significantly higher epidermal NGF expression in PN (5.25 ± 2.49) than in healthy controls (1.09 ± 0.93, p < 0.01) (Figure 1a), whereas BDNF expression remained comparable (Figure S1a). Serum NGF levels did not differ significantly between patients with PN (113.28 ± 143.17 pg/mL) and controls (490.22 ± 703.97 pg/mL), indicating a localized NGF effect in PN pathogenesis (Figure 1a).

Next, we examined how EDN1 regulates NGF expression in the epidermis. Immunofluorescence demonstrated increased NGF expression following EDN1 (100 nM) stimulation in keratinocytes (Figure S1b). Western blot analysis revealed a peak in NGF protein levels at 18 h poststimulation (Figure S1c). siRNA knockdown of EDNRA and EDNRB in HaCaT cells showed that EDN1-induced NGF upregulation was significantly attenuated in EDNRB-deficient cells but remained unaffected by EDNRA knockdown (Figure 1b). These findings indicate that EDNRB, rather than EDNRA, is the key mediator of EDN1-induced NGF expression in keratinocytes.

Given the key role of neuronal pathways in PN, we investigated EDN1's impact on NGF expression in DRG neurons. EDN1 (100 nM) stimulation significantly increased NGF protein expression, peaking at 18 h, as confirmed by western blot (Figure 1c).

Since neurite outgrowth and elongation are critical indicators of neuronal sensitization, we pretreated DRG neurons with a neutralizing NGF antibody (1 μg/mL) and observed a substantial increase in neurite length in the EDN1-treated group (102.61 ± 51.83 μm) compared to controls (65.77 ± 38.38 μm, p < 0.0001), which was attenuated by NGF neutralization (74.01 ± 35.1 μm). The proportion of DRG neurite branching was similarly increased with EDN1 treatment and reduced by NGF blockade (p < 0.05) (Figure 2a). To delineate the receptor-specific effects, DRG neurons were pretreated with selective EDNRA (BQ123) and EDNRB (BQ788) inhibitors. EDN1 treatment significantly increased neurite length (60.7 ± 39 μm, p < 0.01) compared to controls (42.6 ± 27.6 μm). This effect was markedly reduced with EDNRB inhibition (39.8 ± 25.9 μm, p < 0.0001) but not with EDNRA inhibition. A similar pattern was observed in DRG neurite branching (Figure 2b).

We show that EDN1 enhances NGF expression in keratinocytes and DRG neurons, promoting neurite outgrowth. Notably, EDNRB, but not EDNRA, regulates this pathway, underscoring its role in peripheral nerve remodeling. These findings suggest that EDN1-induced NGF upregulation contributes to neural modulation in PN. Moreover, this signaling cascade highlights distinct roles of EDNRB and EDNRA in physiology. Targeting EDNRB may provide a novel therapeutic strategy to interrupt the chronic itch-scratch cycle by blocking EDN1-NGF signaling.

Lai-San Wong: conceptualization (lead), formal analysis (lead), writing – original draft (lead), writing – review and editing (equal). Jenq-Lin Yang: conceptualization (supporting), funding acquisition (equal), writing – original draft (supporting), writing – review and editing (equal). Yu-Ta Yen: investigation (equal), writing – original draft (supporting), writing – review and editing (equal). Chih-Hung Lee: conceptualization (supporting), funding acquisition (equal), supervision (equal), writing – original draft (supporting), writing – review and editing (equal). Jen-Hau Yang: conceptualization (supporting), formal analysis (supporting), investigation (equal), supervision (equal), writing – original draft (supporting), writing – review and editing (equal).

The authors declare no conflicts of interest.