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- Chamcheu, Jean Christopher, et al.
(author)
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Characterization of immortalized human epidermolysis bullosa simplex (KRT5) cell lines : trimethylamine N-oxide protects the keratin cytoskeleton against disruptive stress condition
- 2009
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In: Journal of dermatological science (Amsterdam). - : Elsevier. - 0923-1811 .- 1873-569X. ; 53:3, s. 198-206
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Journal article (peer-reviewed)abstract
- BACKGROUND: Epidermolysis bullosa simplex (EBS) is an autosomal inherited mechano-bullous disease, characterized by intraepidermal blistering and skin fragility caused by mutations in the keratin (KRT) 5 or 14 genes. Despite a vast knowledge about the intermediate filament pathology in this disease, the progress in therapy has been slow. Animal models and well-characterized continuous cell culture models of EBS are needed prior to clinical testing. OBJECTIVES: Our aim was to generate immortalized cell lines as an in vitro model for the study of EBS and test a chemical chaperone, trimethylamine N-oxide (TMAO), as a putative novel therapy. METHODS: We generated four immortalized cell lines, two each from an EBS patient with a KRT5-mutation (V186L) and a healthy control, using human papillomavirus 16 (HPV16) E6E7 as transducer. Cell lines were established in serum-free and serum-containing medium and assessed for growth characteristics, keratin expression profiles, ability to differentiate in organotypic cultures, and response to heat stress with and without the presence of TMAO. RESULTS: All cell lines have been expanded >160 population doublings and their cellular characteristics are similar. However, the formation of cytoplasmic keratin filament aggregates in response to heat-shock treatment differed between EBS and normal cell lines. Notably, serum-free established EBS-cell line was most vulnerable to heat shock but both cell lines exhibited significant reduction in the number of keratin aggregates containing cells by TMAO. CONCLUSION: The immortalized cell lines represent a suitable model for studying novel therapies for EBS. TMAO is a promising new agent for future development as a novel EBS therapy.
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- Forsberg, Sofi, et al.
(author)
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Re-epithelialization from human skin explant cultures is promoted by ligand-activated HER3 receptor
- 2010
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In: Journal of dermatological science (Amsterdam). - : Elsevier BV. - 0923-1811 .- 1873-569X. ; 59:1, s. 7-15
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Journal article (peer-reviewed)abstract
- BACKGROUND: Ligand-stimulated epidermal growth factor receptor (EGFR/HER1) plays a fundamental role in skin biology as potent transducer of mitotic and anti-apoptotic stimuli in keratinocytes. In human epidermis, at least two additional EGFR family members--HER2 and HER3--are expressed but their biological functions in normal and diseased human skin remain obscure. OBJECTIVE: Here, we studied the expression and biological impact of HER3 in regenerating human epidermis formed from skin explants adhered to acellular dermis. METHODS: Neoepidermal HER3 expression was examined by quantitative real-time reverse transcriptase polymerase chain reaction, immunohistochemistry and Western blot analysis. The dynamic effect of HER3 receptor stimulation by recombinant heregulin (HRG)-beta1 was assessed by fluorescence imaging of re-epithelialization. RESULTS: In the neoepidermis, HER3 mRNA and protein were detected with activated receptors being immunolocalized at basal and low suprabasal levels. Exogenous HRG-beta1 at 10-20 ng/ml increased the outgrowth rate corresponding to approximately 30% the response of exogenous EGF. The growth-promoting effect of HRG-beta1 was associated with enhanced HER3 phosphorylation, keratinocyte proliferation and thickening of viable neoepidermis whereas blockade of ligand-binding to HER3 delayed the outgrowth process and inhibited both constitutive and ligand-induced HER3 phosphorylation. HER2 antagonism using an anti-dimerization antibody, pertuzumab, impeded the re-epithelialization rate. In addition, a selective HER2 kinase inhibitor, CP654577, downregulated phospho-HER3 expression suggesting that transactivation of kinase-deficient HER3 was accomplished through dimerization with HER2. CONCLUSION: The study emphasizes the central role of EGFR in epidermal renewal and demonstrates that HRG-activated HER3 contributes to the outgrowth process of epidermis in vitro.
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- Gustafsson, Anna, et al.
(author)
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Effect of IFN-γ on the kynurenine/tryptophan ratio in monolayer-cultured keratinocytes and a 3D reconstructed human epidermis model
- 2020
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In: Journal of dermatological science (Amsterdam). - : Japanese Society for Investigative Dermatology. - 0923-1811 .- 1873-569X. ; 99:3, s. 177-184
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Journal article (peer-reviewed)abstract
- BACKGROUND: Interferon-gamma (IFN-γ) represents a potent inducer for keratinocyte inflammatory and immune activation in vitro. Since tryptophan (trp) conversion to kynurenine (kyn) is involved in inflammation, the topical kyn/trp ratio may serve as a biomarker of skin inflammation. However, the trp metabolism in keratinocytes exposed to IFN-γ is not yet fully understood.OBJECTIVE: The aim of this study was to establish a human epidermis model in order to quantify cytokine and kyn/trp secretion from IFN-γ stimulated cells and tissues. Moreover, to compare the cell response of 2D-cultured keratinocytes and the 3D epidermis model.METHODS: Polycarbonate filters were used on which primary keratinocytes could attach and stratify in order to form the typical layers of reconstructed human epidermis (RHE). After IFN-γ treatment, secretion of kyn/trp was measured by high performance liquid chromatography. Gene and protein expression of indoleamine 2,3-dioxygenase 1 (IDO) was analyzed with real-time PCR and immunohistochemistry. The secretion of cytokines was quantified with ELISA.RESULTS: Trp catabolism to kyn was significantly increased (P < 0.01) in the 2D culture in response to IFN-γ treatment. Before kyn secretion, IDO was strongly upregulated (P < 0.001). IFN-γ treatment also increased the secretion of IL-6 and IL-8 from the keratinocytes. In the RHE, IDO was upregulated by IFN-γ, and kyn secretion could be detected. Interestingly, IDO expression was only present in the basal cells of the RHE.CONCLUSION: Our results suggest that IFN-γ acts as an inducer of trp degradation preferentially in undifferentiated keratinocytes, indicated by the IDO expression in the basal layer of the RHE.
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- Karlsson, Teresa, et al.
(author)
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Keratinocyte differentiation induced by calcium, phorbol ester or interferon-γ elicits distinct changes in the retinoid signalling pathways
- 2010
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In: Journal of dermatological science (Amsterdam). - : Elsevier BV. - 0923-1811 .- 1873-569X. ; 57:3, s. 207-213
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Journal article (peer-reviewed)abstract
- Background: Retinoids influence keratinocyte proliferation and differentiation via binding to nuclear retinoic acid receptors (RARα, -γ) and retinoid X receptor α (RXRα). The effect of keratinocyte differentiation on expression of nuclear retinoid receptors and on the conversion of retinol into retinoic acid has not been examined earlier in depth. Objectives: Our aim was to examine the expression of retinoid receptors and a retinoid-regulated gene CRABPII, as well as the metabolism of exogenous [3H]retinol in cultured human keratinocytes induced to differentiate by exposure to either calcium, phorbol 12-myristate 13-acetate (PMA), or interferon-γ (IFNγ). Methods: Normal human keratinocytes were cultured and exposed to differentiation-inducing agents. The mRNA and protein expression of retinoid receptors were examined using real-time PCR and Western blot. [3H]Retinol uptake and metabolism was monitored by HPLC with on-line radioactivity detection. Results: In calcium-exposed cells, increased expression of RARγ and RXRα, enhanced metabolism of [3H]retinol to 3,4-didehydro-RA (ddRA), and an induction of CRABPII mRNA and protein was noted. In contrast, treatment with PMA and IFNγ reduced the RARγ and RXRα protein expression (preventable by the proteasome inhibitor MG132), increased the accumulation of [3H]RA and/or [3H]ddRA in the cells, and changed the CRABPII transcription. Conclusions: Retinoid signalling is profoundly altered upon differentiation of keratinocytes and the effects depend on how cellular differentiation is initiated.
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| 9. |
- Li, Hao, 1984-, et al.
(author)
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Interactions between FATP4 and ichthyin in epidermal lipid processing may provide clues to the pathogenesis of autosomal recessive congenital ichthyosis
- 2013
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In: Journal of dermatological science (Amsterdam). - : Elsevier BV. - 0923-1811 .- 1873-569X. ; 69:3, s. 195-201
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Journal article (peer-reviewed)abstract
- Background: Autosomal recessive congenital ichthyosis (ARCI) is caused by mutations in 10 differentgenes, of which transglutaminase-1 (TGM1) predominates. A rare form is ichthyosis prematuritysyndrome (IPS) caused by mutations in SLC27A4 encoding fatty acid transporter protein 4 (FATP4),believed to be an acyl-CoA synthetase activating long- and very-long-chain FA. Another ARCI is caused bymutations in NIPAL4, coding for ichthyin, which is proposed to be a magnesium transporter or a transmembrane receptor. A possible interaction between FATP4 and ichthyin has not been studied before.Objective:To find common denominators in the pathogenesis of ARCI.Methods:FATP4 and ichthyin were analyzed by immunofluorescence and proximity ligation assay (PLA) in healthy and ARCI patient skin and in in vitro models of ARCI epidermis.Results:Both proteins were expressed in the upper stratum granulosum of normal epidermis and PLA confirmed a close interaction between FATP4 and ichthyin. In IPS skin lacking FATP4 we found reduced ichthyin expression and this finding could be reproduced in organotypic epidermis with siRNA silenced SLC27A4. In contrast, increased FATP4 staining was found in patients with ichthyin (NIPAL4) mutations and in organotypic epidermis with silenced NIPAL4. In patients with TGM1 mutations, the expression of both FATP4 and ichthyin was increased, but the PLA signal was low probably indicating a malfunctioning protein interaction.Conclusion:Our study suggests that FATP4, ichthyin and TGM1 interact in lipid processing essential for maintaining the epidermal barrier function. It is also hypothesized that ichthyin serves as Mg2+-transporter for FATP4 in this process.
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