Interaction of transcription factors in the proliferation and differentiation of keratinocytes

钙蛋白酶是一种普遍存在于细胞内的胞质半胱氨酸蛋白酶。钙蛋白酶的功能是调节胞吐、细胞融合、凋亡、增殖和EGF受体的降解。在成纤维细胞中，EGF受体通过MAP激酶信号通路激活钙蛋白酶。本研究的目的是确定EGF是否激活HaCaT细胞中的calpain，以及激活的calpain是否反过来诱导凋亡，在免疫印迹中，在加入10 nM EGF后6小时，观察到α-spectrin的150 kDa和145 kDa片段，表明calpain对α-spectrin双联体的蛋白水解。此外，在加入EQF后12小时，m-钙蛋白酶减少，但在Western印迹中JL -钙蛋白酶没有减少。因此，我们认为这种减少是m-钙蛋白酶的自溶。我们接下来在荧光计中使用外源性钙蛋白酶底物AC-LLY-AFC分析钙蛋白酶的激活。我们成功地检测到EGF对钙蛋白酶的激活作用（Student配对检验P< 0.01），钙蛋白酶抑制剂I抑制EGF对钙蛋白酶的激活作用。采用荧光显微镜下的Boc法和TUNEL法检测钙蛋白酶和诱导细胞凋亡。Boc测定显示EGF刺激钙蛋白酶活性。钙蛋白酶抑制剂I可阻断荧光显微镜下的荧光强度。TUNEL法检测的阳性细胞数与Boc法检测的阳性细胞数一致，证明EGF激活的钙蛋白酶以钙蛋白酶依赖的方式诱导HaCaT细胞凋亡。EGF通过激活MAP激酶的信号转导，可诱导钙蛋白酶活性和细胞凋亡。EGF可能通过直接调节角质形成细胞内Ca^<2+>浓度而激活calpain，但EGF激活calpain并诱导角质形成细胞凋亡的信号通路仍有待进一步研究。

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.