|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1997 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1996 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Sp1 nuclear levels have been shown to directly correlate with the proliferative state of the cells. It is known that EGF receptor is activated by a series of reaction of phosphorylation, and post-transcriptional modification of Sp1 by phosphorylation and glycosylation has been correlated with Sp1 transcriptional activation in vitro. We therefore studied changes in the Sp1 nuclear levels in a rat pituitary cell line GH4 stimulated by EGF and Okadaic acid which is a phosphatase inhibitor. Using nuclear extracts from GH4 cells treated with l0nM EGF foe at least 16h, Sp1 binding decreased without a significant changes in the gERE1 and 2 complexes. Immunoblot analysis using the nuclear extracts treated with EGF showed that there was a decrease in the nuclear levels of Sp1. Pretreatment with EGF had little effect on the basal activity of the gastrin promoter. However, the the basal activity was decrease in basal activation of mutated gERE and Sp1 element which confer higher basal activity than the gERE.Treatment of the cells with okadaic acid alone resulted in a similar or greater decrease in Sp1 nuclear levels compared with EGF alone. Treatment of the cells with both EGF and okadaic acid had a synergistic effect, further depressing nuclear levels of Sp1. The loss of Sp1 was observed with EGF and augmented by okadaic acid, suggesting that activation of the EGF receptor phosphorylates Sp1 through increased intracellular serine/threonine protein kinase activity.
4. Okadaic AcidのみによってもEGF単独の刺激と同様あるいはそれ以上に,Sp1タンパクレベルを減少し,Okadaic AcidとEGFは同時刺激によりSp1タンパクレベルに対して互いに相乗的に作用し,著明に減少した.