Role of Sp1 in Transcription of Human ATP2A2 Gene in Keratinocytes

Abstract

The ATP2A2 gene encodes Ca2+-dependent ATPase, the dysfunction of which causes Darier disease. In this study, we analyzed the promoter structure of the human ATP2A2 gene using primary normal human keratinocytes (NHK). Reporter assays showed that deletion of -550/-529, -488/-472, -390/-362, or -42/-21 resulted in a significant decrease in human ATP2A2 promoter activity. Electrophoretic mobility shift assay (EMSA) showed that Sp1 is a transcription factor that binds to the -550/-529 and -488/-472 regions of the promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that Sp1, but not Sp3, binds to the promoter region of the ATP2A2 gene in NHK cells in vivo. Knockdown of Sp1 expression by small interfering RNA resulted in a marked reduction in ATP2A2 promoter activity and ATP2A2 mRNA levels in NHK, suggesting that Sp1 positively transactivates the ATP2A2 promoter in NHK. This is early evidence demonstrating that Sp1 plays an important and positive role in ATP2A2 gene expression in NHK in vivo and in vitro.

Cyclooxygenase‐2 inhibition restores ultraviolet B‐induced downregulation of ATP2A2 /SERCA2 in keratinocytes: possible therapeutic approach of cyclooxygenase‐2 inhibition for treatment of Darier disease

“…1a) under conditions of 60–90% transfection efficiency (data not shown, and Takagi et al. 11 ). UVB treatment increased COX‐2 mRNA level to 10‐fold higher than without UVB, which was reduced by ∼70% in the presence of COX‐2 siRNA in a steady state.…”

“…Although the mechanism of ATP2A2 downregulation by PGE 2 has not been revealed, the function of transcription factors may be affected by PGE 2 because COX‐2 suppressed ATP2A2 promoter activity. Previously, we found that the transcription factor Sp1 transactivated the ATP2A2 promoter 11 . The function of Sp1 was affected by Ca 2+ concentration, which regulated the nuclear localization of Sp1 in keratinocytes 10 .…”

“…The mRNA level is represented as the ratio to that of control siRNA without UVB treatment (= 100%) (b) Promoter activity measured by a reporter assay. Basic promoter, pGL3‐Basic; ATP2A2 promoter, pGL3‐Basic carrying −679/+61 of human ATP2A2 gene 11 . Relative luciferase activity is represented as the ratio to that of pGL3‐Basic without any treatments.…”

“…Normal human keratinocytes (NHK) derived from normal newborn foreskin were purchased from Kurabo (Osaka, Japan) and cultured in serum‐free standard medium (Epilife‐KGM ® ; Invitrogen, Tokyo, Japan) as described previously 10,11 . Third‐to‐fourth‐passage keratinocytes in monolayer at 60–80% confluence were used.…”

“…Keratinocyte cells (1 × 10 5 ) were transfected with 2 μg of reporter plasmid generated in our previous study 11 and 250 ng of pRL‐null (Promega, Madison, WI, U.S.A.) as an internal control for transfection efficiency by Fugene6 (Roche Diagnostics, Indianapolis, IN, U.S.A.). After 24 h of culture, cells were harvested and the luciferase activity of each cell lysate was measured using a Dual‐luciferase assay kit (Promega) and a luminescence detector, Micro Lumat Plus (Berthold, Postfach, Germany) as described previously 10,11 …”