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477 Dietary fat- and obesity-sensitive dermal adipocyte PKCβ induction and inflammation cross-talk
ABSTRACTS | Genetic Disease, Gene Regulation and Gene Therapy 474
Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility C Feng1, Z Lin1, S Li2, H Wang1, Y Yang3 and X Tan2 1 Department of Dermatology, Peking University First Hospital, Beijing, Beijing, China, 2 School of Pharmaceutical Science, Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China and 3 Department of Dermatology, Peking University First Hospital. Peking-Tsinghua Center for Life Sciences, Beijing, China Skin integrity is essential for protection from external stress and trauma. Defects in structural proteins such as keratins cause skin fragility, epitomized by epidermolysis bullosa (EB), a life-threatening disorder. Here we show that dominant mutations of KLHL24, encoding a cullin3eRbx1 ubiquitin ligase substrate receptor, cause EB. We have identified start-codon mutations in the KLHL24 gene in five patients with EB. These mutations lead to truncated KLHL24 protein lacking the initial 28 amino acids (KLHL24-DN28). KLHL24-DN28 is more stable than its wild-type counterpart owing to abolished autoubiquitination. We have further identified keratin 14 (KRT14) as a KLHL24 substrate and found that KLHL24-DN28 induces excessive ubiquitination and degradation of KRT14. Using a knock-in mouse model, we have confirmed that the Klhl24 mutations lead to stabilized Klhl24-DN28 and cause Krt14 degradation. Our findings identify a new disease-causing mechanism due to dysregulation of autoubiquitination and open new avenues for the treatment of related disorders.
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Paraoxonase 1 (PON1) L55M and Q192R polymorphisms, lipid profiles and psoriasis A Abraham Hernandez Collazo1, A Alvarado Navarro2 and M Fafutis Morris2 1 Instituto Dermatologico de Jalisco, Zapopan, Jalisco, Mexico and 2 Centro de Investigacio´n en Inmunologı´a y Dermatologı´a, Zapopan, Jalisco, Mexico Paraoxonase 1 is a serum high-density lipoprotein-bound enzyme with antioxidant function. It hydrolyses lipid peroxides, protecting low-density lipoproteins from oxidative modifications. Patients with psoriasis are at greater risk of oxidative stress, which is associated with abnormal plasma lipid metabolism and cardiovascular disease. The association of the PON1 M allele from L55M polymorphism and Q allele from Q192R polymorfphism with lipid profiles and psoriasis was studied. This case-control study consisted of 100 patients with psoriasis and 100 unrelated healthy controls from the population of Western Mexico. The PON1 L55M and Q192R polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism. Serum lipid levels were determined by enzyme assay. The presence of the PON1 55 M allele wasn’t found to be associated with psoriasis; but PON1 192 R allele was found to be associated with psoriasis in our population (OR¼1.59, 1.06- 2.40 CI 95%, p¼0.02), while homozygous phenotypes RR was strongly associated with psoriasis (OR¼3.42, 1.15- 10.1, p¼0.02). Patients with psoriasis with the PON1 192 R (Q/R, R/R) allele had higher VLDL cholesterol (36.91 27.92 vs 27.76 15.45, p¼0.01) and Castelli index (5.02 1.73 vs 4.41 1.68, p¼0.03) but lower HDL cholesterol (40.97 12.49 vs 45.89 13.65, p¼0.02) than control subjects. The PON1 192 R allele is a risk factor for psoriasis in Western Mexico population. Carriers of this allele have higher levels of VLDL and Castelli index and low HDL. These results indicate that PON 1 Q192R polymorphism may play a role in the pathogenesis and progression of psoriasis and its related complications. These data suggest that patients with psoriasis are more susceptible to cardiovascular diseases by oxidative stress.
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Development of an intrinsic skin sensor for blood glucose level with CRISPR-mediated genome editing in epidermal stem cells J Yue, X Wu and X Wu University of Chicago, Chicago, IL Biointegrated sensor can address various challenges in medicine by transmitting a wide variety of biological signals. A tempting possibility that has not been explored before is whether we can take advantage of genome editing technology to transform a small portion of endogenous tissue to an intrinsic and long-lasting sensor for physiological signals. The human skin and the epidermal stem cells of skin have several unique advantages, making them particularly suited for genetic engineering and applications in vivo. In this report, we develop a novel platform for manipulation and transplantation of epidermal stem cells, and present the key evidence that genome-edited skin stem cells can be exploited for continuous monitoring of blood glucose level in vivo. Additionally, by advanced design of genome editing, we develop autologous skin graft that can sense glucose level and deliver therapeutic proteins for diabetes treatment. Our results reveal the clinical potential for skin somatic gene therapy.
Dietary fat- and obesity-sensitive dermal adipocyte PKCb induction and inflammation cross-talk D Mehta1, R Bansode2, A Wang3, W Huang4 and K Deep4 1 University of Cincinnati College of Medicine, Cincinnati, OH, 2 Center for Excellence in Post Harvest Technologies, North Carolina Research Campus, Kannapolis, NC, 3 University of Chicago, Chicago, IL and 4 The Ohio State University College of Medicine, Columbus, OH Obesity aggravates many skin diseases, including psoriasis, cellulitis, and fungal infection. Despite knowledge that dermal adipocytes are intimately associated with the skin microenvironment, diet- or obesity-induced signaling changes in adipocytes and their impact on cutaneous pathophysiology are not well understood. We previously reported that a systemic PKCß deficiency protects from high-fat diet-induced adipose hypertrophy at various anatomical locations. We now report that the PKCß deficiency is also associated with an improvement in the dermal adipocyte inflammation as well as texture and density of the skin hair. To understand the molecular basis of PKCß action, we determined the high-fat diet-PKCß interaction and the impact of PKCß deficiency on adipocyte signaling and metabolism. We found that dietary fat strongly induces expression of adipose PKCß and inflammatory markers in a time-dependent manner. The impact of diet-PKCß signaling on adipocyte inflammatory mediators (I kappa ß kinase phosphorylation, cytokines, adipokines), insulin-sensitivity (insulin receptor substrate-1 phosphorylation), mitochondrial and autophagy regulators (adaptor protein p66shc phosphorylation, LC3-1/2) were also assessed. To dissect adipose-specific contribution of diet-adipocyte PKCß/inflammation axis to immune responses, wound healing and scaring, and hair-follicle growth, we have generated a floxed PKCß mouse model which will allow us to generate an adipocyte-specific PKCß deficiency. The results presented suggest that PKCß is a physiological transducer of dietary lipids and plays a critical role in modulating dermal inflammatory microenvironment.
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Mutation-specific siRNA Knockdown of GJB2 L Potential gene therapy for Keratitis-ichthyosis-deafness Syndrome M Lee, VA Kinsler, SL Hart and W Di UCL GOS ICH, London, England, United Kingdom Mutations in the GJB2 gene, which encodes connexin 26 (Cx26), cause keratitis-ichthyosisdeafness (KID) syndrome. 87% of patients carry a dominant mutation D50N, which is thought to cause a dominant negative effect on the normal allele product. Current treatment is limited to symptomatic care. We hypothesised that a mutation-specific siRNA to selectively suppress the D50N mutant allele may potentially reverse the disease phenotype. We first tested whether suppression of a single Cx26 allele can lead to haplo-insufficiency in human keratinocytes (KCs). CRISPR/Cas9 was used to generate a Cx26+/- KC line. A scrape loading dye transfer assay (SLDT) used for assessing gap junction intercellular communication was performed in Cx26+/- KCs and non-edited Cx26+/+ KCs. No differences in dye transfer were observed between these two cell lines, indicating haplo-sufficiency when one allele is suppressed. A mutation-specific siRNA was developed after extensive optimisation in vitro and the specificity of the siRNA was assessed using HeLa cells lentivirally transduced with Cx26WT-GFP or Cx26D50N-GFP. The GJB2 mRNA level in transduced HeLa cells was evaluated by qRT-PCR and the protein level by flow cytometry. The results showed reduction of w80% GJB2 mutant gene and w60% mutant Cx26 protein, whereas there were no changes in mRNA and protein levels in WT cells. We further evaluated functional recovery of Cx26 in KID patient derived KCs (KID-KCs) following the siRNA treatment using the SLDT. Results showed significantly improved dye transfer in KID-KCs treated with the siRNA, compared to untreated KID-KCs (p < 0.05). There were no differences in normal KCs with or without siRNA treatment. Further functional assessments such as hemichannel activity are under way to confirm the findings. These data suggest that mutation-targeted siRNA therapy is feasible subject to skin delivery, and could be a potential therapy for KID syndrome.
S82 Journal of Investigative Dermatology (2017), Volume 137
Transcriptomics identifies potential novel therapeutic targets in androgenetic alopecia R Dey-Rao University at Buffalo, Buffalo, NY Genome-wide gene expression from bald and haired scalp of the same individuals was studied by microarray analysis to evaluate pathogenic mechanisms underlying the development and progression of androgenetic alopecia (AGA). Although previous genomic studies and linkage analyses at the DNA level offer a context for identifying putative susceptibility loci, they do not address the altered transcriptional framework in the skin that is ultimately responsible for phenotypic expression. Unbiased clustering analyses of our microarray data reveals a “bald pathology” linked gene signature. Pathway-based analyses of the differentially expressed genes (DEGs) associated with AGA identifies perturbed neurological pathways such as oligodendrocyte precursor cell differentiation and myelination, apoptosis, cell proliferation, and WNT signaling as central drivers of the hair loss process. Interactome analysis of this gene set uncovered several known and novel key transcriptional factors including androgen receptor (AR), a known susceptibility locus for AGA. A comparison of our list of DEGs to previously published molecular studies on AGA identified 16 overlapping genes (including FOS, JAG1 and FZD7) that are dysregulated in the same direction. Furthermore, we identified 14 potential transcriptional “hot spots” in which there is an enhanced correlation of significantly altered gene expression at particular chromosomal locations that may be of particular interest for future genetic association studies. Finally, we integrated functional annotations with clinical criteria as well as prior knowledge of underlying genetics to apply an in silico bioinformatics based approach to identify five novel drug-targets (PTGIR, SP1, BCL2, HDAC2 and NR3C1) for exploration as future AGA therapies.
Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility C Feng1, Z Lin1, S Li2, H Wang1, Y Yang3 and X Tan2 1 Department of Dermatology, Peking University First Hospital, Beijing, Beijing, China, 2 School of Pharmaceutical Science, Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China and 3 Department of Dermatology, Peking University First Hospital. Peking-Tsinghua Center for Life Sciences, Beijing, China Skin integrity is essential for protection from external stress and trauma. Defects in structural proteins such as keratins cause skin fragility, epitomized by epidermolysis bullosa (EB), a life-threatening disorder. Here we show that dominant mutations of KLHL24, encoding a cullin3eRbx1 ubiquitin ligase substrate receptor, cause EB. We have identified start-codon mutations in the KLHL24 gene in five patients with EB. These mutations lead to truncated KLHL24 protein lacking the initial 28 amino acids (KLHL24-DN28). KLHL24-DN28 is more stable than its wild-type counterpart owing to abolished autoubiquitination. We have further identified keratin 14 (KRT14) as a KLHL24 substrate and found that KLHL24-DN28 induces excessive ubiquitination and degradation of KRT14. Using a knock-in mouse model, we have confirmed that the Klhl24 mutations lead to stabilized Klhl24-DN28 and cause Krt14 degradation. Our findings identify a new disease-causing mechanism due to dysregulation of autoubiquitination and open new avenues for the treatment of related disorders.
476
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Paraoxonase 1 (PON1) L55M and Q192R polymorphisms, lipid profiles and psoriasis A Abraham Hernandez Collazo1, A Alvarado Navarro2 and M Fafutis Morris2 1 Instituto Dermatologico de Jalisco, Zapopan, Jalisco, Mexico and 2 Centro de Investigacio´n en Inmunologı´a y Dermatologı´a, Zapopan, Jalisco, Mexico Paraoxonase 1 is a serum high-density lipoprotein-bound enzyme with antioxidant function. It hydrolyses lipid peroxides, protecting low-density lipoproteins from oxidative modifications. Patients with psoriasis are at greater risk of oxidative stress, which is associated with abnormal plasma lipid metabolism and cardiovascular disease. The association of the PON1 M allele from L55M polymorphism and Q allele from Q192R polymorfphism with lipid profiles and psoriasis was studied. This case-control study consisted of 100 patients with psoriasis and 100 unrelated healthy controls from the population of Western Mexico. The PON1 L55M and Q192R polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism. Serum lipid levels were determined by enzyme assay. The presence of the PON1 55 M allele wasn’t found to be associated with psoriasis; but PON1 192 R allele was found to be associated with psoriasis in our population (OR¼1.59, 1.06- 2.40 CI 95%, p¼0.02), while homozygous phenotypes RR was strongly associated with psoriasis (OR¼3.42, 1.15- 10.1, p¼0.02). Patients with psoriasis with the PON1 192 R (Q/R, R/R) allele had higher VLDL cholesterol (36.91 27.92 vs 27.76 15.45, p¼0.01) and Castelli index (5.02 1.73 vs 4.41 1.68, p¼0.03) but lower HDL cholesterol (40.97 12.49 vs 45.89 13.65, p¼0.02) than control subjects. The PON1 192 R allele is a risk factor for psoriasis in Western Mexico population. Carriers of this allele have higher levels of VLDL and Castelli index and low HDL. These results indicate that PON 1 Q192R polymorphism may play a role in the pathogenesis and progression of psoriasis and its related complications. These data suggest that patients with psoriasis are more susceptible to cardiovascular diseases by oxidative stress.
477
Development of an intrinsic skin sensor for blood glucose level with CRISPR-mediated genome editing in epidermal stem cells J Yue, X Wu and X Wu University of Chicago, Chicago, IL Biointegrated sensor can address various challenges in medicine by transmitting a wide variety of biological signals. A tempting possibility that has not been explored before is whether we can take advantage of genome editing technology to transform a small portion of endogenous tissue to an intrinsic and long-lasting sensor for physiological signals. The human skin and the epidermal stem cells of skin have several unique advantages, making them particularly suited for genetic engineering and applications in vivo. In this report, we develop a novel platform for manipulation and transplantation of epidermal stem cells, and present the key evidence that genome-edited skin stem cells can be exploited for continuous monitoring of blood glucose level in vivo. Additionally, by advanced design of genome editing, we develop autologous skin graft that can sense glucose level and deliver therapeutic proteins for diabetes treatment. Our results reveal the clinical potential for skin somatic gene therapy.
Dietary fat- and obesity-sensitive dermal adipocyte PKCb induction and inflammation cross-talk D Mehta1, R Bansode2, A Wang3, W Huang4 and K Deep4 1 University of Cincinnati College of Medicine, Cincinnati, OH, 2 Center for Excellence in Post Harvest Technologies, North Carolina Research Campus, Kannapolis, NC, 3 University of Chicago, Chicago, IL and 4 The Ohio State University College of Medicine, Columbus, OH Obesity aggravates many skin diseases, including psoriasis, cellulitis, and fungal infection. Despite knowledge that dermal adipocytes are intimately associated with the skin microenvironment, diet- or obesity-induced signaling changes in adipocytes and their impact on cutaneous pathophysiology are not well understood. We previously reported that a systemic PKCß deficiency protects from high-fat diet-induced adipose hypertrophy at various anatomical locations. We now report that the PKCß deficiency is also associated with an improvement in the dermal adipocyte inflammation as well as texture and density of the skin hair. To understand the molecular basis of PKCß action, we determined the high-fat diet-PKCß interaction and the impact of PKCß deficiency on adipocyte signaling and metabolism. We found that dietary fat strongly induces expression of adipose PKCß and inflammatory markers in a time-dependent manner. The impact of diet-PKCß signaling on adipocyte inflammatory mediators (I kappa ß kinase phosphorylation, cytokines, adipokines), insulin-sensitivity (insulin receptor substrate-1 phosphorylation), mitochondrial and autophagy regulators (adaptor protein p66shc phosphorylation, LC3-1/2) were also assessed. To dissect adipose-specific contribution of diet-adipocyte PKCß/inflammation axis to immune responses, wound healing and scaring, and hair-follicle growth, we have generated a floxed PKCß mouse model which will allow us to generate an adipocyte-specific PKCß deficiency. The results presented suggest that PKCß is a physiological transducer of dietary lipids and plays a critical role in modulating dermal inflammatory microenvironment.
478
479
Mutation-specific siRNA Knockdown of GJB2 L Potential gene therapy for Keratitis-ichthyosis-deafness Syndrome M Lee, VA Kinsler, SL Hart and W Di UCL GOS ICH, London, England, United Kingdom Mutations in the GJB2 gene, which encodes connexin 26 (Cx26), cause keratitis-ichthyosisdeafness (KID) syndrome. 87% of patients carry a dominant mutation D50N, which is thought to cause a dominant negative effect on the normal allele product. Current treatment is limited to symptomatic care. We hypothesised that a mutation-specific siRNA to selectively suppress the D50N mutant allele may potentially reverse the disease phenotype. We first tested whether suppression of a single Cx26 allele can lead to haplo-insufficiency in human keratinocytes (KCs). CRISPR/Cas9 was used to generate a Cx26+/- KC line. A scrape loading dye transfer assay (SLDT) used for assessing gap junction intercellular communication was performed in Cx26+/- KCs and non-edited Cx26+/+ KCs. No differences in dye transfer were observed between these two cell lines, indicating haplo-sufficiency when one allele is suppressed. A mutation-specific siRNA was developed after extensive optimisation in vitro and the specificity of the siRNA was assessed using HeLa cells lentivirally transduced with Cx26WT-GFP or Cx26D50N-GFP. The GJB2 mRNA level in transduced HeLa cells was evaluated by qRT-PCR and the protein level by flow cytometry. The results showed reduction of w80% GJB2 mutant gene and w60% mutant Cx26 protein, whereas there were no changes in mRNA and protein levels in WT cells. We further evaluated functional recovery of Cx26 in KID patient derived KCs (KID-KCs) following the siRNA treatment using the SLDT. Results showed significantly improved dye transfer in KID-KCs treated with the siRNA, compared to untreated KID-KCs (p < 0.05). There were no differences in normal KCs with or without siRNA treatment. Further functional assessments such as hemichannel activity are under way to confirm the findings. These data suggest that mutation-targeted siRNA therapy is feasible subject to skin delivery, and could be a potential therapy for KID syndrome.
S82 Journal of Investigative Dermatology (2017), Volume 137
Transcriptomics identifies potential novel therapeutic targets in androgenetic alopecia R Dey-Rao University at Buffalo, Buffalo, NY Genome-wide gene expression from bald and haired scalp of the same individuals was studied by microarray analysis to evaluate pathogenic mechanisms underlying the development and progression of androgenetic alopecia (AGA). Although previous genomic studies and linkage analyses at the DNA level offer a context for identifying putative susceptibility loci, they do not address the altered transcriptional framework in the skin that is ultimately responsible for phenotypic expression. Unbiased clustering analyses of our microarray data reveals a “bald pathology” linked gene signature. Pathway-based analyses of the differentially expressed genes (DEGs) associated with AGA identifies perturbed neurological pathways such as oligodendrocyte precursor cell differentiation and myelination, apoptosis, cell proliferation, and WNT signaling as central drivers of the hair loss process. Interactome analysis of this gene set uncovered several known and novel key transcriptional factors including androgen receptor (AR), a known susceptibility locus for AGA. A comparison of our list of DEGs to previously published molecular studies on AGA identified 16 overlapping genes (including FOS, JAG1 and FZD7) that are dysregulated in the same direction. Furthermore, we identified 14 potential transcriptional “hot spots” in which there is an enhanced correlation of significantly altered gene expression at particular chromosomal locations that may be of particular interest for future genetic association studies. Finally, we integrated functional annotations with clinical criteria as well as prior knowledge of underlying genetics to apply an in silico bioinformatics based approach to identify five novel drug-targets (PTGIR, SP1, BCL2, HDAC2 and NR3C1) for exploration as future AGA therapies.