|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 2003 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 2002 : ¥1,800,000 (Direct Cost : ¥1,800,000)
A gene expression profiling of mouse genome was done by using a GeneChip【○!R】 oligonucleotide microarray (Affymetrix) and mRNA extracted from epidermal growth factor (EGF)-stimulated NIH3T3 cells. The GADD45 gamma and GADD45 beta were identified to be up-and down-regulated immediately after 30 min of EGF administration. Therefore we attempted to clarify the mechanism of GADD45 gene expression regulated by EGF stimulation.
RT-PCR analysis, RNase protection assay and Western blot analysis demonstrated that GADD45 alpha mRNA/protein, and GADD45 gamma mRNA/ protein expression were increased after 30 min of EGF administration. In contrast, GADD45 beta mRNA/ protein expression were decreased within 15 min of EGF administration. To investigate a relationship between these phenomena and MAP kinase (MAPK)/ Extracellular signal-regulated kinase (ERK) signaling pathway, GADD45 mRNA and proteins expression were analyzed in the presence of a specific MEK inhibitor PD08059 (20 M). No significant increase or decrease of known GADD45 mRNA/ proteins expression was observed.
To understand the crosstalk of JNK and /or p38 MAP kinase in EGF signaling pathway, we performed in vitro kinase assay by using specific antibody for JNK and p238 MAP kinase. After 15 min of EGF administration, JNK was activated (2.2 fold), which in turn inactivated after 30-120 min. However, p38 MAPK activation was not seen after 0-120 min of EGF administration.
To investigate a relationship between JNK activity and GADD45 beta in EGF signaling pathway, we measured JNK activity in the presence of siRNA for GADD45 beta. interestingly, JNK activity was increased (3-6 fold) after 0-60 min of EGF administration. These results suggest that GADD45 beta might be involved in JNK signaling under EGF stimulation.