Insights into molecular and functional mechanisms behind inherited heart and skin disorders.

PhDDesmosomes are macromolecular, dynamic and adaptable complexes that connect intermediate filaments of neighboring cells in a variety of tissues, generating a large mechanically resilient structure. The importance of maintaining desmosome homeostasis for tissue integrity and optimal organ function has been revealed through the identification of desmosome-associated disorders and mechanistic studies into desmosome regulation. This thesis focuses on inherited skin and heart conditions linked to mutations in desmosomal genes or in genes believed to be implicated in desmosome regulation. Part of this thesis is focused on the molecular analysis and identification of novel desmosomal mutations in patients clinically diagnosed with Arrhythmogenic Right Ventricular Cardiomyopathy, and the genetic diagnosis of patients with hypotrichosis, hypotrichosis and PPK or acral peeling skin syndrome. Patients were analysed using a number of different genetic techniques including custom capture array, HaloPlex targeted resequencing, exome capture and Sanger sequencing. Both novel and previously reported mutations were identified in DSP, DSC2, DSG2, PKP2, DSG4 or CSTA in patients diagnosed with these disorders. The molecular mechanisms behind mutations in the protease inhibitors cystatin A and calpastatin, leading to the skin disorders exfoliative ichthyosis and PLACK syndrome, were also investigated. In vitro analysis, using siRNA-mediated knockdown in the immortalised keratinocyte cell line HaCaT, demonstrated that these mutations, affecting the structure and function of the protease inhibitors, lead to deficient intercellular adhesion, possibly through the indirect regulation of desmosomal complexes through their target proteases

Cell Cycle- and Cancer-Associated Gene Networks Activated by Dsg2: Evidence of Cystatin A Deregulation and a Potential Role in Cell-Cell Adhesion

This work was supported by grants from the National Institutes of Health (Mahoney, R01AR056067; Riobo, RO1 GM088256). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Modulation of CSTA expression by Dsg2.

<p>(A) A431 cells were treated for 72 hr with 100 nM of scrambled RNA or <i>Dsg2</i> siRNA. Western blot analysis for Dsg2 and cystatin A shows that knockdown of Dsg2 reduced CSTA level. Immunoblotting for Actin showed equal loading. (B) The Western blot results were quantified and expression level of each band was normalized against Actin. Values shown are percentage of expression against control untreated. The results showed significant reduction in Dsg2 in response to <i>Dsg2</i> siRNA but not scrambled siRNA, while knockdown of Dsg2 slightly reduced the expression of CSTA. The change was statistically significant. Bar = mean ± s.e.m. *p<0.05 and ***p<0.001 using Student’s <i>t</i> test. (C) A431 cells were stably transfected with shRNA to GFP (shGFP) or Dsg2 (shDsg2) and selected in puromycin. Immunoblotting showed loss of Dsg2 reduced CSTA expression in the shDsg2 cells, as compared to the shGFP cells. Actin was used as a loading control. (D) Quantification of the Western blot results showed reduction in Dsg2 in the shDsg2 cell as compared to the shGFP cells and knockdown of Dsg2 reduced CSTA expression. Bar = mean ± s.e.m. *p<0.05 and ***p<0.001 using Student’s <i>t</i> test. E) Immunofluorescence of Dsg2 and CSTA showed that knockdown of Dsg2 reduced the expression of Dsg2 and CSTA in the A431-shDsg2 as compared to A431-shGFP cells. Nuclei were counter-stained with DAPI (blue).</p

Dsg2 enhances cystatin A expression <i>in vivo</i>.

<p>(A) Western blot analysis of skin lysates from 3 newborn and 3 adult C57Bl6 mice shows high expression of Csta in newborn but virtually undetectable in adult skin. Actin was used as a control for equal loading. (B) Immunofluorescent staining confirms the Western blotting results showing high level of Csta in newborn wild-type mouse skin. Enlarged image in inset shows cytoplasmic as well as nuclear staining for Csta. (C) Western analysis for Dsg2 and Csta in adult wild-type and Inv-Dsg2 transgenic mouse skin. The results showed expression of the Flag-tagged Dsg2 and Csta in the transgenic but not wild-type mice. Actin showed equal loading. (D) Immunofluorescence was performed on adult skin of wild-type and transgenic mice revealing increased levels CSTA in transgenic skin. Nuclei were counter-stained with DAPI (blue).</p

Modulation of cell adhesion by CSTA and Dsg2.

<p>(A) Immunofluorescence of normal human skin (A) and palm (B) showing low levels of Dsg2 in the basal layer (arrows) of the normal skin (inset: enlarged image) but high levels in both the basal and differentiated layers (arrow head) in the palm. Note: Immunostaining was performed at the same time and images were captured at the same exposure. (B) A431-shGFP and A431-shDsg2 cells were treated with scrambled RNA or siRNA to <i>CSTA</i> for 72 hr and then subjected to the <i>in vitro</i> mechanical stress dispase-based dissociation assay. Bright field images showing loss of Dsg2 or CSTA induced fragmentation and the loss of both had a synergistic effect on cell adhesion. (C) Graph showing the number of fragments for each condition. i, shGFP; ii, shGFP + scrRNA; iii, shGFP + siCSTA; iv, shDsg2; v, shDsg2 + scrRNA; vi, shDsg2 + siCSTA. Bar = mean ± s.e.m. *p< 0.05 and **p<0.01 using Student’s <i>t</i> test.</p

Loss of CSTA leads to destabilized intercellular connections.

<p>Cells were treated with non-targeting pool scrRNA or with <i>CSTA</i> siRNA (CSTA KD) followed by mechanical stretching for 4 hr. Cells were allowed to adhere, fixed, and immunostained for Dsg2 (A) and cytokeratin 14 (B) or lysed in Laemmli buffer and immunoblotted for desmoplakin (C). Knockdown of CSTA in keratinocytes resulted in cytoplasmic relocalization of Dsg2, breakage of cytokeratin intercellular connections, and loss of the desmosomal protein, desmoplakin.</p

Top ten genes up- or down-regulated in response to Dsg2.

<p>Top ten genes up- or down-regulated in response to Dsg2.</p

Differential gene expression between Inv-Dsg2 transgenic and wild-type mouse skin.

<p>(A) Total RNA was isolated from the skin of 2 wild-type and 2 Inv-Dsg2 transgenic mice, reverse transcribed, biotin-labeled and applied to a mouse cDNA microarray. The dendogram (heat map) shows that 492 genes were either up-regulated (red/orange) or down-regulated (blue/green) in transgenic (T1 and T2) and control (W1 and W2) mice. (B) Volcano plot shows the log2 (fold change) in x-axis versus the—log10 (p value) in the y-axis. The points having a fold-change less than 2 (log2 = 1) are shown in gray. The vertical green lines demarcate where the fold change equals 2 (right line) or equals—2 (left line). The horizontal green line demarcates where the p value is 0.05, with points above the line having p<0.05 and points below the line having p>0.05. Depicted in red are the genes that exhibit a greater than 2 fold change with a p>0.05 in transgenic epidermis as compared to control. The arrows indicate genes of interest. (C) Quantitative real-time RT-PCR analysis reveals an average of 34.33±1.32 fold increase in Dsg2 RNA expression in Inv-Dsg2 transgenic (Tg) compared to that of wild-type (WT). In addition, RNA expression for transgenic relative to control were: <i>Csta1</i>, 113.24 ±2.23; <i>Csta2</i>, 1227.04 ±1.26; <i>Csta2l1</i>, 1.11 ±0.47; <i>Csta3</i>, 26.97±0.44 (Bar = mean ± s.d.; (*p< 0.05; **p<0.01; ***p<0.001; Student’s <i>t</i> test).</p

Differentially expressed genes in response to Dsg2.

<p>Differentially expressed genes in response to Dsg2.</p