Reduction in keratin aggregates in epidermolysis bullosa simplex keratinocytes after pretreatment with trimethylamine N‐oxide

Epidermolysis bullosa simplex (EBS) is a dominantly inherited skin disease caused by mutations in the keratin 5 (KRT5) or KRT14 genes 1. Some reports suggested that fever and/or hot weather may exacerbate EBS phenotype 2. Effective EBS therapies are still lacking. Molecular chaperones are proteins whose main function is to promote the correct folding of polypeptides (s1). Molecules such as trimethylamine N-oxide (TMAO) and sodium 4-phenylbutyrate (4-PBA) act as chemical chaperones (s2) with protein folding and stabilization activities (s3, s4, s5). Treatment of affected epidermal cells by chemical chaperones to correct the misfolded and aggregated keratins that characterize EBS seems a viable therapeutic option 3. Furthermore, the type I keratins K16 and K17 polymerize with K5, and upregulation of these proteins could replace the mutant K14 in the heterodimer and improve disease pathology (s6). Hence, chemical chaperones which can reduce keratin aggregates formation and upregulate K16 and K17 in EBS-affected cells would be ideal therapeutic candidates for EBS. Investigate the potential therapeutic effects of two chemical chaperones, TMAO and 4-PBA, in the treatment of EBS by comparing keratin cytoskeleton abnormalities in cells of patients with EBS and non-EBS participants in a heat-shock model, an ideal model as patients with EBS usually suffer from disease exacerbation in a hot and humid climate 2. See supplementary information. To determine whether TMAO or 4-PBA had a beneficial effect on EBS keratinocytes, cytoplasmic keratin aggregates were evaluated by immunofluorescence. Unstressed or heat-stressed control cells showed very few or no keratin aggregates (Fig. 1a, b). Of the four cell lines, EBS21 cells had the highest increase in the percentage of cells with aggregates (42.5%) after heat shock (Fig. S1). EBS21 keratinocytes showed keratin aggregates in resting conditions (Fig. 1c), and transient heat shock led to a collapse of the keratin cytoskeleton and an increase in keratin aggregates (Fig. 1d). Pretreatment of EBS21 cells with TMAO at 150mM prior to heat shock reduced keratin aggregation (Fig. 1e). Percentage of cells with aggregates decreased from 42.5% (after heat shock) to 19.5% (P = 0.03). Replication assays showed a significant reduction in cells with aggregates in EBS10 (P = 0.002), but not in EBS1 (Fig. S1). Non-significant aggregates reduction was observed in the three EBS cells with pretreatment of 4-PBA at 10mm (data not shown). Protein levels of Hsp70 and Hsp40 were assessed by Western blot to determine whether pretreatment with TMAO affected their levels in EBS21 and control cells. Our data revealed that Hsp70 was 5.5-fold more expressed in unstressed condition in EBS21 compared with control cells and that Hsp40 was not induced (Fig. 2). Heat shock or pretreatment with TMAO alone had no effect on Hsp70 or Hsp40 levels. Pretreatment with TMAO in heat-shocked EBS21 cells reduced Hsp70 and Hsp40 expression significantly compared with unstressed EBS21 cells (P = 0.03 and P = 0.05, respectively) (Fig. 2b, d). Pretreatment of TMAO in unstressed EBS21 cells did not significantly change KRT16 and KRT17 gene expression from those without pretreatment. Similar results were observed for control cells (data not shown). These data demonstrated a promising potential for TMAO as a therapeutic agent for patients with EBS. In this study, TMAO (150mM) partially reduced the percentage of heat-shocked EBS cells with aggregates, although it was not able to eliminate all aggregates. TMAO may prevent aggregate formation by restoring misfolded proteins 4 or may promote protein degradation of misfolded proteins through the ubiquitin-proteasome pathway 5. The increase in Hsp70 protein expression observed in unstressed EBS21 as compared to control cells may be due to other genetic variations than the KRT14 mutation. However, the reduction in Hsp70 and Hsp40 protein level by TMAO and heat shock was not observed in control cells, suggesting that the mutation is associated with an environment in which TMAO functions beneficially. Our data of TMAO reducing Hsp70 and Hsp40 protein levels are in line with results observed in red blood cells 6. A reduced expression of molecular chaperones by TMAO in cells under stress may be explained by a preventing effect of TMAO from heat shock damages, reducing the need to elicit the cellular heat-shock response 6. In contrast to our findings, TMAO treatment at 50 and 100mm in two cell lines with KRT5 mutations had been shown to induce moderate upregulation of Hsp70 protein expression 5. These differences suggest that the mechanism of action of TMAO may be dependent on the mutations of the affected cells 7, 8 or on the concentration of TMAO 5. KRT16 and KRT17 gene expression results showed no differences in contrast to the increase in K16 and K17 observed in a sulforaphane-treated K14-null mice 9. Maybe gene knock-out will elicit more pronounced biological consequences than a mutated gene. Although TMAO demonstrated itself as a promising therapeutic candidate for EBS with its thermo stress protective role, particularly in those with mutations in KRT5 and KRT14, it is worthy to mention that reduction in heat-induced aggregates in EBS cells by TMAO could simply be with no therapeutic value and no potential benefit to patients with EBS. Further mechanistic studies are needed to relate keratin aggregates to stronger and more resilient skin. We thank the participants in EBS biobank. This work was supported by Le Groupe Riverin Inc and by La Fondation du Grand défi Pierre Lavoie. MB contributed to experiment design, performed the experiments with JL, TF and ML. MB and JL wrote the manuscript. JP, CM, AD and CM performed skin biopsies. VLG contributed to study design and manuscript. CL leads the research programme, built the biobank, designed and supervised the study and manuscript redaction. All participants provided written informed consents. The study was approved by the ethics committee from the Hôpital Sainte-Justine and from the Centre intégré universitaire de santé et de services sociaux du Saguenay–Lac-Saint-Jean. The Declaration of Helsinki protocols were followed. No conflict of interest to declare. Data S1. Material and methods Table S1. Clinical status and mutations found in three EBS cases Additional references - S1–S11. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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