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Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation.

van den Bogaard EH, Podolsky MA, Smits JP, Cui X, John C, Gowda K, Desai D, Amin SG, Schalkwijk J, Perdew GH, Glick AB - J. Invest. Dermatol. (2015)

Bottom Line: Using transcriptome analysis of Ahr(-/-) and Ahr(+/+) murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation.Primary Ahr(-/-) keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr(+/+) keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM) SGA360.Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.

ABSTRACT
Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr(-/-) and Ahr(+/+) murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr(-/-) keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr(+/+) keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM) SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression, and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology.

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Reduced terminal differentiation protein expression caused by AHR inactivationHuman skin equivalents were generated using de-epidermised dermis and expression of keratin 10 (KRT10), filaggrin (FLG) and loricrin (LOR) was followed in time by harvesting the skin equivalents directly after submerged culture, and after 4, 6 and 10 days of air-liquid interface culture (each block represents one day of culture). Treatment with GNF351 (500 nM, arrows) was initiated at day 6 and sustained until day 10 of air-liquid interface culture. Magnification inlays show epidermal differentiation protein expression affected by AHR inactivation. (n= 2 keratinocytes donors). Scale bar = 100 μm.
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Figure 4: Reduced terminal differentiation protein expression caused by AHR inactivationHuman skin equivalents were generated using de-epidermised dermis and expression of keratin 10 (KRT10), filaggrin (FLG) and loricrin (LOR) was followed in time by harvesting the skin equivalents directly after submerged culture, and after 4, 6 and 10 days of air-liquid interface culture (each block represents one day of culture). Treatment with GNF351 (500 nM, arrows) was initiated at day 6 and sustained until day 10 of air-liquid interface culture. Magnification inlays show epidermal differentiation protein expression affected by AHR inactivation. (n= 2 keratinocytes donors). Scale bar = 100 μm.

Mentions: To further examine the effect of AHR antagonists on epidermal differentiation we generated epidermal skin equivalents using human primary keratinocytes cultured on plastic inert filters. We tested the effect of antagonists added at different time points during generation of the human skin equivalents. When the keratinocytes were in submerged culture (proliferation/attachment phase) or when monolayers were initially brought to the air-liquid interface, addition of GNF351 or CH223191 substantially suppressed the stratification process and formation of the stratum corneum (Figure 3a). Expression of late differentiation markers involucrin and filaggrin was strongly reduced, but the early differentiation marker keratin 10 was less affected (Figure 3b). Addition of antagonists during the last phase of air-liquid interface culture (from day 4 or 7 onwards) resulted in thinning of the stratum corneum but did not affect involucrin or filaggrin expression (Figure 3b). However, when skin equivalents were generated using de-epidermized dermis, treatment with GNF351 4 days after transfer to air-liquid interface reduced the expression and number of cell layers expressing loricrin and filaggrin, while expression of keratin 10 was delayed (Figure 4). Since AHR antagonists were added during the proliferation phase of the skin equivalent development (submerged culture), we tested if they affected keratinocyte proliferation. There was a significant reduction in the percentage of Ki67 positive cells and cell number after treating proliferating monolayer cultures of human keratinocytes with AHR antagonists for 48 h (Figure 5, S4). In contrast, skin equivalents generated on inert filters and treated with GNF351 during the submerged phase or at day one of transfer to the air-liquid interphase had more Ki67 positive basal cells at the end of the skin equivalent development compared to untreated cultures (Figure 3b).


Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation.

van den Bogaard EH, Podolsky MA, Smits JP, Cui X, John C, Gowda K, Desai D, Amin SG, Schalkwijk J, Perdew GH, Glick AB - J. Invest. Dermatol. (2015)

Reduced terminal differentiation protein expression caused by AHR inactivationHuman skin equivalents were generated using de-epidermised dermis and expression of keratin 10 (KRT10), filaggrin (FLG) and loricrin (LOR) was followed in time by harvesting the skin equivalents directly after submerged culture, and after 4, 6 and 10 days of air-liquid interface culture (each block represents one day of culture). Treatment with GNF351 (500 nM, arrows) was initiated at day 6 and sustained until day 10 of air-liquid interface culture. Magnification inlays show epidermal differentiation protein expression affected by AHR inactivation. (n= 2 keratinocytes donors). Scale bar = 100 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402116&req=5

Figure 4: Reduced terminal differentiation protein expression caused by AHR inactivationHuman skin equivalents were generated using de-epidermised dermis and expression of keratin 10 (KRT10), filaggrin (FLG) and loricrin (LOR) was followed in time by harvesting the skin equivalents directly after submerged culture, and after 4, 6 and 10 days of air-liquid interface culture (each block represents one day of culture). Treatment with GNF351 (500 nM, arrows) was initiated at day 6 and sustained until day 10 of air-liquid interface culture. Magnification inlays show epidermal differentiation protein expression affected by AHR inactivation. (n= 2 keratinocytes donors). Scale bar = 100 μm.
Mentions: To further examine the effect of AHR antagonists on epidermal differentiation we generated epidermal skin equivalents using human primary keratinocytes cultured on plastic inert filters. We tested the effect of antagonists added at different time points during generation of the human skin equivalents. When the keratinocytes were in submerged culture (proliferation/attachment phase) or when monolayers were initially brought to the air-liquid interface, addition of GNF351 or CH223191 substantially suppressed the stratification process and formation of the stratum corneum (Figure 3a). Expression of late differentiation markers involucrin and filaggrin was strongly reduced, but the early differentiation marker keratin 10 was less affected (Figure 3b). Addition of antagonists during the last phase of air-liquid interface culture (from day 4 or 7 onwards) resulted in thinning of the stratum corneum but did not affect involucrin or filaggrin expression (Figure 3b). However, when skin equivalents were generated using de-epidermized dermis, treatment with GNF351 4 days after transfer to air-liquid interface reduced the expression and number of cell layers expressing loricrin and filaggrin, while expression of keratin 10 was delayed (Figure 4). Since AHR antagonists were added during the proliferation phase of the skin equivalent development (submerged culture), we tested if they affected keratinocyte proliferation. There was a significant reduction in the percentage of Ki67 positive cells and cell number after treating proliferating monolayer cultures of human keratinocytes with AHR antagonists for 48 h (Figure 5, S4). In contrast, skin equivalents generated on inert filters and treated with GNF351 during the submerged phase or at day one of transfer to the air-liquid interphase had more Ki67 positive basal cells at the end of the skin equivalent development compared to untreated cultures (Figure 3b).

Bottom Line: Using transcriptome analysis of Ahr(-/-) and Ahr(+/+) murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation.Primary Ahr(-/-) keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr(+/+) keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM) SGA360.Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.

ABSTRACT
Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr(-/-) and Ahr(+/+) murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr(-/-) keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr(+/+) keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM) SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression, and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology.

Show MeSH
Related in: MedlinePlus