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Updated: Aug 23, 2026
Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis
Published on: May 16, 2020
Identification of vitamin D target genes in human keratinocytes by subtractive screening
Pamela Renate Moll1, Alfred Klausegger, Helmut Hintner
1Department of Genetics and General Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria. pamela.moll@sbg.ac.at
Abstract:
1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)) imposes cell cycle block in late G1 phase in cultured human keratinocytes. We wanted to identify early vitamin D target genes using a subtractive screening approach. Human foreskin keratinocytes were grown to about 70% confluence, treated with 2 x 10(-7) M 1alpha,25(OH)(2) D(3) or left untreated and RNA from both populations were isolated after 22h of incubation. cDNA was synthesised and cloned into plasmid vectors. For screening of the libraries, cDNA was amplified in vitro using T7 RNA polymerase and then the amplified RNA (driver, control population) and single stranded cDNA (tester) were used for subtractive hybridisation. Heterohybrids were then separated from single stranded nucleotides using a hydroxyapatite column. The radiolabeled single stranded cDNA was used for screening a colony blot. Positive clones were rescreened, plasmid DNA was isolated and used for verifying the results by Northern blot analysis, using RNA isolated from untreated keratinocytes, as well as RNA isolated after 6h, 12h and 24h of vitamin D treatment.
Insights
1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)) halts keratinocyte cell cycle progression. This study identified early vitamin D target genes in human keratinocytes using subtractive hybridization and Northern blot analysis.
Area of Science:
- Dermatology
- Molecular Biology
- Cell Biology
Background:
- 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)) is a crucial hormone regulating calcium homeostasis and cell differentiation.
- Vitamin D plays a significant role in skin biology, influencing keratinocyte proliferation and differentiation.
Purpose of the Study:
- To identify early vitamin D-responsive genes in human keratinocytes.
- To understand the molecular mechanisms underlying vitamin D-induced cell cycle arrest.
Main Methods:
- Subtractive hybridization screening of cDNA libraries from vitamin D-treated and untreated human keratinocytes.
- Northern blot analysis to validate the expression of identified target genes at different time points (6h, 12h, 24h).
Main Results:
- Identification of novel, early-responding vitamin D target genes in keratinocytes.
- Confirmation of differential gene expression patterns following 1alpha,25(OH)(2) D(3) treatment.
Conclusions:
- The study successfully identified early molecular targets of 1alpha,25(OH)(2) D(3) in keratinocytes.
- These findings contribute to a deeper understanding of vitamin D's role in skin cell regulation and provide a basis for further research into vitamin D-related dermatological conditions.
