Alvey, L, The Metabolic Role of CARD14 in Epithelial Barrier Dysfunction
From Lauren Alvey
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Introduction: Atopic dermatitis is an “entry point” for subsequent development of food allergy and asthma in children. A GWAS in children with atopic dermatitis identified the rs11652075 missense variant (R820W) in the caspase recruitment domain family member 14 (CARD14) gene to be associated with reduced expression levels of the barrier protein filaggrin, suggesting it may negatively impact barrier function. We recently found that the variant is associated with decreased expression of the crucial transcription factor MYC as well as the dysregulation of numerous mitochondrial genes. Given the prominent role of MYC in both cellular metabolism and skin barrier formation, we thus hypothesized that the reduced MYC in keratinocytes harboring the CARD14 variant mediates mitochondrial dysregulation thus predisposing skin barrier dysfunction.
Methods: Homozygous WT HaCaT keratinocytes and HaCaT cells that have been CRISPR edited to carry homozygous R820W CARD14 alleles were used in all experiments. RNA-sequencing and subsequent bioinformatics analyses compared expression of known MYC regulated mitochondrial genes between these cell lines. Utilizing 10058-F4, a known inhibitor of MYC, we examined MYC’s genotype-dependent impact on the expression of MYC target genes through qPCR, as well as mitochondrial mass (using the stain NAO) and mitochondrial membrane potential (using the stain TMRE) through live cell microscopy. Further, Real Time ATP Production Rate Assays (also known as Seahorse) were conducted in WT and R820W cells with and without 10058-F4 treatment to examine mitochondrial respiration rates.
Results: Specific analysis of RNA-sequencing results for MYC-regulated mitochondrial genes identified downregulation of BNIP3 mRNA in R820W keratinocytes compared to WT. BNIP3 is a crucial protein for mitochondrial turnover that is known to be regulated by MYC in many cell lines, but this relationship has not been studied in keratinocytes. qPCR confirmed that BNIP3 expression is decreased in R820W cells compared to WT (p=0.015); however, 10058-F4 treatment did not rescue BNIP3 mRNA levels. Preliminary microscopy studies indicated decreased mitochondrial mass with 10058-F4 treatment but no difference between genotypes. When normalized by mitochondrial mass, there was no difference in mitochondrial membrane potential between genotypes or with 10058-F4 treatment. The Seahorse assay showed no difference in mitochondrial ATP production rates between cell lines or with 10058-F4 treatment.
Conclusions: Our findings indicate that there is differential expression of BNIP3 in CARD14 variant cells, but that this effect is independent of MYC. Our data also suggest that MYC activity supports mitochondrial mass (which agrees with previously published literature), but that MYC does not directly affect mitochondrial membrane potential in keratinocytes. Further, our data did not show evidence of the R820W variant affecting mitochondrial mass, mitochondrial membrane potential, or mitochondrial ATP production, suggesting that CARD14 does not significantly contribute to mitochondrial function in keratinocytes.
Methods: Homozygous WT HaCaT keratinocytes and HaCaT cells that have been CRISPR edited to carry homozygous R820W CARD14 alleles were used in all experiments. RNA-sequencing and subsequent bioinformatics analyses compared expression of known MYC regulated mitochondrial genes between these cell lines. Utilizing 10058-F4, a known inhibitor of MYC, we examined MYC’s genotype-dependent impact on the expression of MYC target genes through qPCR, as well as mitochondrial mass (using the stain NAO) and mitochondrial membrane potential (using the stain TMRE) through live cell microscopy. Further, Real Time ATP Production Rate Assays (also known as Seahorse) were conducted in WT and R820W cells with and without 10058-F4 treatment to examine mitochondrial respiration rates.
Results: Specific analysis of RNA-sequencing results for MYC-regulated mitochondrial genes identified downregulation of BNIP3 mRNA in R820W keratinocytes compared to WT. BNIP3 is a crucial protein for mitochondrial turnover that is known to be regulated by MYC in many cell lines, but this relationship has not been studied in keratinocytes. qPCR confirmed that BNIP3 expression is decreased in R820W cells compared to WT (p=0.015); however, 10058-F4 treatment did not rescue BNIP3 mRNA levels. Preliminary microscopy studies indicated decreased mitochondrial mass with 10058-F4 treatment but no difference between genotypes. When normalized by mitochondrial mass, there was no difference in mitochondrial membrane potential between genotypes or with 10058-F4 treatment. The Seahorse assay showed no difference in mitochondrial ATP production rates between cell lines or with 10058-F4 treatment.
Conclusions: Our findings indicate that there is differential expression of BNIP3 in CARD14 variant cells, but that this effect is independent of MYC. Our data also suggest that MYC activity supports mitochondrial mass (which agrees with previously published literature), but that MYC does not directly affect mitochondrial membrane potential in keratinocytes. Further, our data did not show evidence of the R820W variant affecting mitochondrial mass, mitochondrial membrane potential, or mitochondrial ATP production, suggesting that CARD14 does not significantly contribute to mitochondrial function in keratinocytes.
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