J Korean Med Sci.  2010 Apr;25(4):619-625. 10.3346/jkms.2010.25.4.619.

Expression Profiling of Calcium Induced Genes in Cultured Human Keratinocytes

• Lee JS
• Kim MR
• Kim NS
• Kim YS
• Yang JM
• Cho AY
• Lee Y
• Kim CD
• Lee JH

• KMID: 1792935
• DOI: http://doi.org/10.3346/jkms.2010.25.4.619

Abstract

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. To examine the gene expression profile in calcium-induced keratinocyte differentiation, we constructed a normalized cDNA library using mRNA isolated from these calcium-treated keratinocytes. After sequencing about 10,000 clones, we were able to obtain 4,104 independent genes. They consisted of 3,699 annotated genes and 405 expressed sequence tags (ESTs). Some were the genes involved in constituting epidermal structures and others were unknown genes that are probably associated with keratinocytes. In particular, we were able to identify genes located at the chromosome 1q21, the locus for the epidermal differentiation complex, and 19q13.1, another probable locus for epidermal differentiation-related gene clusters. One EST located at the chromosome 19q13.1 showed increased expression by calcium treatment, suggesting a novel candidate gene relevant to keratinocyte differentiation. These results demonstrate the complexity of the transcriptional profile of keratinocytes, providing important clues on which to base further investigations of the molecular events underlying keratinocyte differentiation.

Keyword

Keratinocyte Differentiation; Calcium; Normalized Library; Gene Expression Profiling

MeSH Terms

Calcium/*metabolism
Cells, Cultured
Chromosome Mapping
Chromosomes, Human
Expressed Sequence Tags
Gene Expression Profiling
*Gene Expression Regulation
Gene Library
Humans
Keratinocytes/cytology/*physiology
Molecular Sequence Data
Oligonucleotide Array Sequence Analysis
Calcium

Figure

• 

  Fig. 1 Schematic overview of the normalized cDNA library construction. cDNAs were prepared using mRNA isolated from keratinocytes cultured under 1.2 mM calcium, then cloned into pT7T3-Pac vector. After making single-stranded library using helper phage, subtraction was performed against driver cDNAs. The remaining single-stranded DNAs were purified by HAP chromatography, converted into double-stranded plasmids, then used for making normalized library.

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  Fig. 2 Chromosomal localization of the genes obtained from normalized library. Location was determined using Ensembl Genome Browser. Underlined gray numbers indicate chromosome numbers. Gene numbers and percentages from normalized library are shown under each chromosome.

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  Fig. 3 Expressed sequence tags (ESTs) and known genes in epidermal differentiation complex (EDC) on chromosome 1q21 (A) and 19q13.1 (B). EST genes identified from normalized library are shown in bold (denoted by arrow).

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  Fig. 4 Expression of selected expressed sequence tags (ESTs) in keratinocytes cultured in vitro. Total RNAs were extracted from the keratinocytes cultured under 1.2 mM calcium at the indicated time points. Two mg of total RNAs were reverse transcribed and then used for PCR amplification. (A) ESTs located on chromosome 1q21, (B) ESTs located on chromosome 19q13.1. Cyclophilin is used as an internal control.

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