| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Calpain is an ubiquitous intracellular cytoplasmic cysteine protease. The function of calpain is to regulate exocytosis, cell fusion, apoptosis, proliferation and the degradation of EGF receptors. Activation of calpain through EGF receptor occurs via MAP kinase signaling pathway in fibroblast. The purpose of this study is to determine whether EGF activates calpain in HaCaT cells, and whether the activated calpain in turn induces apoptosis.
In immunoblotting, both 150 kDa and 145 kDa fragments of α-spectrin were observed six hours after the addition of 10 nM EGF, indicated proteolysis of α-spectrin doublet by calpain. Also, m-calpain decreased 12 h after the addition of EQF, but JL -calpain did not decrease in western blotting. So, we regarded the decrease as autolysis of m-calpain.
We next analyzed the activation of calpain using exogenous calpain substrate, AC-LLY-AFC in a fluorometer. We succeeded in detecting the activation of calpain by EGF( P< 0.01 by Student's paired test), which was inhibited by calpain inhibitor I. To detect calpain and apoptotic assay in induviual cells, we adopted Boc assay and TUNEL assay using fluorescent microscope. The Boc assay showed that EGF stimulated calpain activity. The intensity of fluorescence microscopy was blocked by calpain inhibitor I. The positive cells in TUNEL assay were consistent with those of Boc assay, proving that activated calpain by EGF induced apoptosis in a calpain-dependent manner.
We detected calpain activity and apoptosis with high concentratitons of EGF in HaCaT cells. Activation of signal transduction via MAP kinase by EGF may induce calpain activity and apoptosis. Perhaps EGF may modulate Ca^<2+> concentration directly and induce activation of calpain in keratinocyte.
It remains the subject of a future study to determine by which signal pathway EGF activates calpain and induces apoptosis in keratinocytes.
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