Finding a treatment for X-linked Recessive Ichthyosis Sofia Miettinen Supervisor – Edel O’Toole Experimental Pathology iBSc 2018/19 Centre for Cell Biology and Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry
12
Embed
Finding a treatment for X-linked Recessive Ichthyosis ... · Sofia Miettinen Supervisor – Edel O’Toole Experimental Pathology iBSc 2018/19 Centre for Cell Biology and Cutaneous
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
activity under calcium shift conditions for CR-KO1 compared to CR-WT (p <0.05), figure 1.A.
Western blot showed a decrease in JunB in the nuclear fraction of CR-KO1 compared to CR-
WT, figure 1.B. JunB expression in 3D organotypic models of XLRI skin grown from CR-
WT and CR-KO1 cell lines was significantly reduced in CR-KO1 cell lines compared to CR-
WT (p < 0.01), figure 1.C and 1.D.
As JunB expression was significantly decreased, DSigDB was used to select drug treatments
that upregulate AP-1 expression: celecoxib, cholecalciferol, and quercetin were selected (3).
Haematoxylin and Eosin staining, figure 2, showed that the three drug treatments all
decreased epidermal thickness.
GAPDH
JunB
1 0.4
D C B A
Figure 1. Comparison of XLRI 3D organotypic models in CRISPR knockout (CR-KO)
and wildtype (CR-WT)
(A). Activator protein 1 (AP-1) expression in CR-KO1 keratinocyte cell line compared to CR-WT
keratinocyte cell line measured with dual luciferase assay with *P0.05 (B). JunB expression was
analyzed by western blot. GAPDH was used as an internal control. (C). Representative images of
immunofluorescence (IF) staining of JunB (red) in CR-WT and CR-KO1 3D organotypics. DAPI
(blue) was used as a nuclear stain. (D). Intensity of JunB was measured using ImageJ, the average
intensity per cell was calculated and shown as a percentage of CR-WT organotypic. Data represents
meanSD with **P0.01
Figure 2 – H&E staining of drug treated CR-WT and CR-KO1 cell line 3D organotypic models Haematoxylin and eosin staining was performed on 3D organotypic models grown from CR-WT and CR-KO1
cell lines. Images were taken from the thickest portion of the epidermis. Scale bar = 200 μm.
XLRI patients have increased polar lipids throughout the epidermis, and so treatment aims to
decrease the polar lipids content. All three drug treatments decreased polar lipids in the CR-
KO1 cell lines compared to the CR-KO1 negative control, figures 3-5.
JunB protein expression was validated as significantly decreased in CR-KO1 cell lines. As seen
in figure 6, JunB expression was increased by similar amounts by all concentrations of drug
treatments in the CR-KO1 3D models compared to CR-KO1 negative control.
Figure 3 – Nile red staining of celecoxib treated CR-WT and CR-KO1 3D organotypic models
3D organotypic model cultures grown using CR-WT and CR-KO1 cell lines were treated with celecoxib 0.05
μM and 0.5 μM. Nile red was added to CR-WT and CR-KO1 negative controls (NC) and drug treated 3D
organotypic cultures. Red = polar lipids, green = non-polar lipids. Scale bar = 100 μm.
Figure 4 – Nile red staining of cholecalciferol treated CR-WT and CR-KO1 3D organotypic models
3D organotypic model cultures grown using CR-WT and CR-KO1 cell lines were treated with
cholecalciferol 0.01 μM and 0.1 μM. Nile red was added to CR-WT and CR-KO1 negative controls (NC)
and drug treated 3D organotypic cultures. Red = polar lipids, green = non-polar lipids. Scale bar = 100 μm.
Figure 5 – Nile red staining of quercetin treated CR-WT and CR-KO1 3D organotypic models
3D organotypic model cultures grown using CR-WT and CR-KO1 cell lines were treated with quercetin 0.05
μM and 0.5 μM. Nile red was added to CR-WT and CR-KO1 negative controls (NC) and drug treated 3D
organotypic cultures. Red = polar lipids, green = non-polar lipids. Scale bar = 100 μm.
Figure 6 – JunB expression in celecoxib, cholecalciferol and quercetin treated CR-WT and CR-KO1
3D organotypic models
3D organotypic model culture grown using CR-WT and CR-KO1 cell lines tissue was stained for JunB. Blue
staining = nucleus, red staining = JunB. Scale bar = 100 μm.
Conclusion:
With the AP-1 subunits forming a complex cascade that can be altered by numerous changes,
it makes identifying the cause of the epidermal changes in XLRI difficult. From our data so
far, it is speculated that decreased JunB activity could be causing the changes.
XLRI has limited treatment options so far, with current treatment not targeting specific
pathways. A further understanding of exact pathways affected by STS gene knockout could
open novel treatment options. This work so far has suggested some interesting pathways for
follow-up to improve the treatment of XLRI.
References:
(1) Oji V, Tadini G, Akiyama M, Blanchet Bardon C, Bodemer C, Bourrat E, et al.
Revised nomenclature and classification of inherited ichthyoses: Results of the First
Ichthyosis Consensus Conference in Sorèze 2009. J Am Acad Dermatol. 2010 Oct
1;63(4):607–41.
(2) van Dam H, Castellazzi M. Distinct roles of Jun : Fos and Jun : ATF dimers in
oncogenesis. Oncogene. 2001 Apr 8;20(19):2453–64.
(3) Yoo M, Shin J, Kim J, Ryall KA, Lee K, Lee S, et al. DSigDB: drug signatures
database for gene set analysis. Bioinformatics. 2015 Sep 15;31(18):3069–71.