Globotriaosylceramide-induced Reduction of KCa1.1 Channel Activity and Activation of the Notch1 Signaling Pathway in Skin Fibroblasts of Male Fabry Patients with Pain.

Background: Fabry disease (FD) is an X-linked lysosomal storage disorder that leads to cellular globotriaosylceramide (Gb3) accumulation due to mutations in the gene encoding alpha-galactosidase A. Trigger-induced acral burning pain is an early FD symptom of unknown pathophysiology. We aimed at investigating the potential role of skin fibroblasts in nociceptor sensitization. Patients and methods: We enrolled 40 adult FD patients and ten healthy controls, who underwent a 6-mm skin punch biopsy at the lower leg. Dermal fibroblasts were cultivated and analyzed for Gb3 load. Fibroblast electrical activity was assessed using patch-clamp analysis at baseline and upon incubation with agalsidase-alpha for 24 h. We investigated gene expression of C-C motif chemokine ligand 2 (CCL2), Ca(2+)activated K+-channel 1.1 (K(Ca)1.1), interferone-gamma (IFN-gamma), transforming growth factor-beta 1 (TGF-beta 1), and transmembrane receptor notch homolog 1 (Notch1) using quantitative real-time-PCR, and protein levels of K(Ca)1.1 by ELISA. Gene expression was determined at baseline and after fibroblast stimulation with tumor necrosis factor-alpha (TNF), modeling inflammation as a common pain trigger in FD. Results: Total Gb3 load was higher in FD fibroblasts than in control fibroblasts (p < .01). Upon increase of intracellular Ca2+ concentrations, we detected differential electrical activity of K(Ca)1.1 in fibroblasts obtained from patients with FD. Gene expression (p < .05) and protein levels of K(Ca)1.1 (p < .05) were higher in fibroblasts from FD patients compared to control fibroblasts, whereas electric channel activity was lower in FD fibroblasts. After incubation with agalsidase-alpha, we observed an over-proportionate increase of K(Ca)1.1 activity in FD fibroblasts reaching 7-fold the currents of control cells (p < .01). Gene expression studies revealed higher mRNA levels of CCL2, INF-gamma, and Notch1 in FD fibroblasts compared to controls at baseline and after TNF incubation (p < .05 each), while TGF-beta 1 was higher in FD fibroblasts only after incubation with TNF (p < .05). Conclusions: Gb3 deposition in skin fibroblasts may impair K(Ca)1.1 activity and activate the Notch1 signaling pathway. The resulting increase in pro-inflammatory mediator expression may contribute to cutaneous nociceptor sensitization as a potential mechanism of FD-associated pain.