Analysis of calcium-inducible genes in keratinocytes using suppression subtractive hybridization and cDNA microarray.
Terminal differentiation of skin keratinocytes is a vertically directed multistep process that is tightly controlled by the sequential expression of a variety of genes. To gain further insight into the molecular events involved in this process, we used suppression subtraction hybridization (SSH) and cDNA microarray analysis. Messenger RNAs were isolated from primary skin keratinocytes cultured in vitro after treatment with calcium and then SSH was performed. A total of 840 cDNA clones were obtained from subtracted libraries, and these cDNA clones were used to make the microarray slides. Time-course cDNA microarray analysis (1, 3, 7, and 14 days after calcium treatment) revealed the global gene expression profile during keratinocyte differentiation. Of the 840 genes tested, 290 showed a greater than twofold change in expression level at least once over four time points. The genes were clustered into six groups according to their expression pattern using self-organizing map analysis and showed the global feature of function-related regulation. The genes related to keratinocyte differentiation were markedly up-regulated by calcium treatment. In addition, a unique pattern of increase was seen in the expression of genes related to ribosomal proteins. On the other hand, transcripts involved in metabolism, DNA repair, transcription, and translation were generally down-regulated. These results demonstrate the complexity of the gene expression profile that contributes to the spatiotemporal regulation of keratinocyte differentiation.
Seo
Eun-Young
EY
Department of Dermatology, School of Medicine, Chungnam National University, 640 Daesa-dong, Daejeon 301-040, Korea.
Namkung
Jung-Hyun
JH
Lee
Kyu-Mi
KM
Lee
Woong-Hee
WH
Im
Myung
M
Kee
Sun-Ho
SH
Tae Park
Geon
G
Yang
Jun-Mo
JM
Seo
Young-Joon
YJ
Park
Jang-Kyu
JK
Deok Kim
Chang
C
Lee
Jeung-Hoon
JH
D002118:Calcium / Q000502:physiology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D004260:DNA Repair; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D006801:Humans; D017403:In Situ Hybridization; D015603:Keratinocytes / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic; D015854:Up-Regulation
D012333:RNA, Messenger; D002118:Calcium