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- Zhang, Hanqian
(författare)
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Congenital Recessive Ichthyosis : Studies of Gene Expressions Related to Keratinocyte Differentiation and Skin Barrier Repair
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Autosomal recessive congenital ichthyosis (ARCI) is a rare monogenetic disorder characterized by a defective skin barrier, hyperkeratosis, and dry, scaly skin. It affects keratinocyte differentiation and is caused by mutations in any of at least 12 genes believed to control the formation of ω-O-acylceramide and the corneocyte lipid envelope (CLE): ABCA12, ALOXE3, ALOX12B, CERS3, CYP4F22, ELOVL4, LIPN, NIPAL4, PNPLA1, SDR9C7, SLC27A4, and TGM1.Studies of keratinocyte differentiation and gene expression in ARCI may help us better understand the pathobiology of skin barrier formation. One way to verify that ARCI-related gene products are operating in a chain of events essential for lipid barrier formation is to use immunofluorescence and in situ proximity ligation assays to demonstrate the proteins’ colocalizations in the epidermis. In paper I, a new method for the objective quantitative image analysis of protein expression and colocalization in different epidermal layers of skin sections was developed using free, open-source software, CellProfiler. Using this method and microarray analyses of skin biopsies from ARCI patients with TGM1 mutations (n = 5) compared with those of healthy controls (n = 4), many ARCI-related genes were found to be upregulated in patient epidermis (paper II). Because many other genes involved in keratinocyte differentiation and immune/inflammatory response, including PPARD, were also induced in the patients’ microarrays, the effects of a ligand-dependent transcription factor, PPARδ, encoded by PPARD, were studied on ARCI-related gene expression in cultured keratinocytes, usually showing the pronounced upregulation by PPARδ agonists (paper III). Furthermore, using previous array data obtained from cultured differentiated keratinocytes and from skin biopsies of patients with TGM1 mutations, nine novel candidate markers of differentiation were identified, and the upregulation was verified by qPCR of mRNA from cultured keratinocytes and skin biopsies. These transcripts were also induced by PPARδ agonists in cultured proliferating keratinocytes (paper IV).To conclude, the upregulation of other ARCI-related genes in patients with TGM1 mutations might reflect a feedback mechanism in ω-O-acylceramide biosynthesis, which, however, is unable to restore the patients’ skin barrier. In theory, substitution therapy with ω-O-acylceramide and recombinant TGm-1 may be required. Because PPARδ activation appears involved in upregulating ARCI-related genes and nine novel differentiation marker genes, all potentially important for barrier repair, this approach could become a treatment option for several types of ichthyosis and wound healing.
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- Zhang, Hanqian, et al.
(författare)
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Identification of novel gene products involved in epidermal keratinocyte differentiation and pathologic skin barrier repair
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A functional skin barrier relies on a correct structural organization of the outermost epidermal layer, stratum corneum, as the final stage of keratinocyte terminal differentiation. However, the factors involved in keratinocyte differentiation, especially those which are important for the skin barrier function and barrier repair, are still largely unknown. In this study we re-analysed two sets of microarrays obtained from cultured differentiated keratinocytes or skin biopsies of autosomal recessive congenital ichthyosis (ARCI) patients with hyperkeratosis and defect skin barrier due to TGM1 mutations. When comparing these arrays to those of proliferating keratinocytes and healthy control epidermis respectively, we identified 24 augmented genes not previously associated with keratinocyte differentiation and skin barrier repair. Increased expression of 9 of these genes, i.e. AKR1B10, BLNK, ENDOU, GCNT4, GLTP, RHCG, SLC15A1, TMEM86A, VSNL1, was verified by qPCR of differentiated keratinocytes and patients skin extracts. In a separate experiment, the regulation of the genes by two well-known transcription factors, retinoic acid receptors (RARs) and peroxisome proliferator activating receptors (PPARs), was studied in cell cultures by adding the pan-RAR agonist all-trans retinoic acid (atRA) or PPARd agonist GW501516. In line with the anti-keratinizing effects of retinoids, atRA reduced the mRNA expressions of some of these genes both in monolayer and organotypic keratinocyte cultures. Confirming the importance of PPARd for keratinocyte differentiation, PPARd-agonist GW501516 induced all 9 novel genes associated with differentiation and skin barrier repair. The results increase the understanding about genes involved in human epidermal keratinocyte differentiation, with potential repercussions on the development of new ways to restore the barrier function in conditions with a defect skin barrier.
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- Zhang, Hanqian, et al.
(författare)
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Patients with congenital ichthyosis and TGM1 mutations overexpress other ARCI genes in the skin : Part of a barrier repair response?
- 2019
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Ingår i: Experimental dermatology. - : John Wiley & Sons. - 0906-6705 .- 1600-0625. ; 28:10, s. 1164-1171
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Tidskriftsartikel (refereegranskat)abstract
- Autosomal recessive congenital ichthyosis (ARCI) is a group of monogenic skin disorders caused by mutations in any of at least 12 different genes, many of which are involved in the epidermal synthesis of ω-O-acylceramides (acylCer). AcylCer are essential precursors of the corneocyte lipid envelope crosslinked by transglutaminase-1 (TGm-1), or a yet unidentified enzyme, for normal skin barrier formation. We hypothesized that inactivating TGM1 mutations will lead to a compensatory overexpression of the transcripts involved in skin barrier repair, including many other ARCI-causing genes. Using microarray we examined the global mRNA expression profile in skin biopsies from five ARCI-patients with TGM1 mutations and four healthy controls. There were a total of 599 significantly differentially expressed genes (adjusted P<0.05), out of which 272 showed more than 1.5 log2fold-change (FC) up- or down-regulation. Functional classification of the latter group of transcripts showed enrichment of mRNA encoding proteins mainly associated with biological pathways involved in keratinocyte differentiation and immune response. Moreover, the expression of seven out of twelve ARCI-causing genes were significantly increased (FC=0.98-2.05). Also, many of the genes involved in keratinocyte differentiation (cornified envelope formation) and immune response (anti-microbial peptides and proinflammatory cytokines) were upregulated. The results from the microarray analysis were also verified for selected genes at the mRNA level by qPCR and at the protein level by semi-quantitative immunofluorescence. The upregulation of these genes might reflect a compensatory induction of acylCer biosynthesis as a part of a global barrier repair response in the patient´s epidermis.
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- Zhang, Hanqian, et al.
(författare)
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PPARδ agonists upregulate transcripts in the ω-O-acylceramide pathway essential for the formation of corneocyte lipid envelopes in epidermis and pathogenic in ichthyosis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- At least twelve proteins are involved in the biosynthesis and transport of ω-O-acylceramide (acylCer), a lipid which is covalently attached to the cornified cell envelope of epidermal keratinocytes essential for a proper permeability barrier. Deleterious mutations in any of the involved genes may cause autosomal recessive congenital ichthyosis (ARCI). The regulation of mRNA transcripts involved in the acylCer pathway is sparsely studied although it is known that retinoids reduce the expression of most of these transcripts and that peroxisome proliferator-activated receptor-d (PPARd) agonists induce the expression of at least three genes (ABCA12, CERS3 and ELOVL4). In this study, we examined the effect of several PPAR agonists on the mRNA expression of twelve ARCI-related genes in cultured human epidermal keratinocytes. In short, PPARd agonists, but not PPARa- and PPARg-agonists, markedly induced the genes after 24 hours exposure, and the induction was more pronounced in pre-confluent proliferating cells as compared to post-confluent differentiated cells, although the latter cells showed a much higher baseline expression. For eight of the genes the induction by PPARd-agonist GW501516 could be abolished by pre-treatment with PPARd-antagonist GSK0660. Reassuringly, GW501516 induced the same genes also in human epidermal equivalents. These results suggest that ligand activation of PPARd should be tested for its ability to restore a defect skin barrier caused by an insufficient production of acylCer.
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- Zhang, Hanqian, et al.
(författare)
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Quantitative image analysis of protein expression and colocalisation in skin sections
- 2018
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Ingår i: Experimental dermatology. - : Wiley. - 0906-6705 .- 1600-0625. ; 27:2, s. 196-199
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Tidskriftsartikel (refereegranskat)abstract
- Immunofluorescence (IF) and in situ proximity ligation assay (isPLA) are techniques that are used for in situ protein expression and colocalisation analysis, respectively. However, an efficient quantitative method to analyse both IF and isPLA staining on skin sections is lacking. Therefore, we developed a new method for semi-automatic quantitative layer-by-layer measurement of protein expression and colocalisation in skin sections using the free open-source software CellProfiler. As a proof of principle, IF and isPLA of ichthyosis-related proteins TGm-1 and SDR9C7 were examined. The results indicate that this new method can be used for protein expression and colocalisation analysis in skin sections.
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