Identification of calcium-induced genes in HaCaT keratinocytes by polymerase chain reaction-based subtractive hybridization.
Suppression subtractive hybridization, a PCR-based method for cDNA subtraction, was used to identify differentially expressed genes in keratinocytes. Differentiation was induced by elevating the calcium level in the cell culture medium. Using HaCaT immortalized keratinocytes cultured in the presence of a high calcium concentration, we isolated 60 clones representing 48 different genes. By reverse Northern analysis, 13 genes were scored as overexpressed in these HaCaT cells. Northern blot analysis was used to confirm differential gene expression. Six genes, keratin 1, plasminogen activator inhibitor type 2 (PAI-2), ferritin H, peroxiredoxin 5 (PRDX5), insulin-like growth factor binding protein-3 (IGFBP-3), and one EST gene, were differentially expressed in HaCaT cells cultured in the presence of a high calcium concentration. Two of these genes, keratin 1 and PAI-2, are differentially expressed during keratinocyte terminal differentiation. IGFBP-3, which has reduced expression during epidermal differentiation, was increased after culture in a high-calcium medium for 2 or 5 days. Overexpression of the ferritin H and PRDX5 genes due to elevated calcium has not been reported in keratinocytes. We demonstrated the expression of IGFBP-3, ferritin H, PRDX5, and one gene of a matching sequence from the EST database during differentiation in primary cultured normal human keratinocytes. The EST gene expressed two transcripts of 1.8 kb and 2.5 kb in HaCaT cells, and the transcripts were confirmed to increase in keratinocytes cultured in a high-calcium medium.
Seo
Eun-Young
EY
Department of Dermatology, Chungnam University Hospital, Daesa-dong 640, Jung-gu, Daejeon 301-040, South Korea.
Piao
Yong-Jun
YJ
Kim
Jeong-Soo
JS
Suhr
Ki-Beom
KB
Park
Jang-Kyu
JK
Lee
Jeung-Hoon
JH
D002118:Calcium / Q000008:administration & dosage*; D002454:Cell Differentiation / Q000502:physiology; D002461:Cell Line, Transformed; D003470:Culture Media / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005293:Ferritins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D007633:Keratins / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009994:Osmolar Concentration; D017396:Plasminogen Activator Inhibitor 2 / Q000235:genetics; D016133:Polymerase Chain Reaction
D003470:Culture Media; D018972:Insulin-Like Growth Factor Binding Protein 3; D017396:Plasminogen Activator Inhibitor 2; D007633:Keratins; D005293:Ferritins; D002118:Calcium