Deciphering the calcitriol-induced transcriptomic response in keratinocytes: presentation of novel target genes

Abstract

Numerous studies to date have been aimed at unraveling the large suite of calcitriol (1a,25-dihydroxyvitamin D 3 ) response genes in diverse tissues including skin, where this hormone is involved in regulating keratinocyte proliferation, differentiation, permeability barrier formation, innate immunity promotion, antimicrobial peptide production, and wound healing. However, the various approaches differ considerably in probed cell types, scale, throughput, and statistical reliability and do, of note, not reveal much overlap. To further expand our knowledge on presently elusive targets and characterize the extent of fragmentation of existing datasets, we have performed whole-transcriptome microarray examinations of calcitriol-treated human primary keratinocytes. Out of 28 869 genes investigated, we uncovered 86 differentially expressed (67 upregulated and 19 downregulated) candidates that were functionally clustered into five annotation categories: response to wounding, protease inhibition, secondary metabolite biosynthesis, cellular migration, and amine biosynthetic processes. A complementary RTq-PCR study of 78 nominees selected thereof demonstrated significant differential expression of 55 genes (48 upregulated and seven downregulated) within biological replicates. Our hit list contains nine previously authenticated targets (16.36%, proof of concept) and 46 novel genes (83.6%) that have not yet been explicitly described as being differentially regulated within human primary keratinocytes. Direct vitamin D receptor response element predictions within the regulatory promoter regions of 50 of the RTq-PCR-validated targets agreed with known biological functionality and corroborated our stringent data validation pipeline. Altogether, our results indicate the value of continuing these kinds of gene expression studies, which contribute to an enhanced comprehension of calcitriol-mediated processes that may be dysregulated in human skin pathophysiology.

Vitamin D and the RNA transcriptome: more than mRNA regulation

“…This particular study from White (Akutsu et al, 2001) and co-workers identified 38 genes that were responsive to 1α,25(OH) 2 D 3 exposure, which represented approximately 1% of the transcriptome studied, and included GADD45A . These earlier studies already suggested at the footprint of the VDR dependent transcriptome (reviewed in Rid et al, 2013). In many ways these studies highlighted the heterogeneity of VDR actions that was to be identified subsequently by ChIP-Seq studies.…”

Vitamin D Receptor and RXR in the Post‐Genomic Era

“…Similarly, studies in squamous models, following 1α,25(OH) 2 D 3 exposure, identified various targets included GADD45A (Akutsu et al, 2001b) which was confirmed by others in different cell types (Akutsu et al, ; Jiang et al, ; Khanim et al, ). These early studies suggested central functions at the footprint of the VDR dependent transcriptome (reviewed in [Rid et al, ]) including cell cycle regulation (Akutsu et al, ; Palmer et al, ; Eelen et al, ; Wang et al, 2005b). In many ways, these studies also highlighted the heterogeneity of VDR actions that was to be identified subsequently by ChIP‐Seq studies.…”

Whole-transcriptome gene expression profiling in an epidermolysis bullosa simplex Dowling-Meara model keratinocyte cell line uncovered novel, potential therapeutic targets and affected pathways

“…A further example from our list of dysregulated genes is the smallest known acidic keratin, KRT19 (significantly down-regulated in our microarray analysis, independently confirmed by both qRT-PCR and western blotting). In our previous study, in which we applied a similar whole-transcriptome microarray methodology to calcitriol-stimulated human primary keratinocytes, we [ 41 ] found a significantly increased expression level of KRT19 after treatment with the indicated secosteroid. Hence, irrespective of the exact role of KRT19 in the disease mechanism, it could constitute another useful biomarker in EBS.…”