Interaction of transcription factors in the proliferation and differentiation of keratinocytes

钙蛋白酶是一种普遍存在于胞浆内的半胱氨酸蛋白酶。钙蛋白酶的功能是调节胞吐、细胞融合、细胞凋亡、增殖和EGF受体的降解。在成纤维细胞中，通过EGF受体激活钙蛋白酶是通过MAPK信号通路实现的。本研究的目的是确定表皮生长因子是否激活HaCaT细胞中的钙蛋白，以及激活的钙蛋白是否诱导细胞凋亡。在免疫印迹中，加入10 nM的表皮生长因子6小时后，观察到150 kDa和145 kDa的α-血影蛋白片段，这表明α-血影蛋白偶联被钙蛋白降解。免疫印迹显示，加入EQF后12h，m-calain表达降低，而JL-calain表达无明显变化。接下来，我们在荧光仪上分析了外源钙蛋白底物AC-LLY-AFC对钙蛋白的激活作用。我们成功地检测到了EGF对Calain的激活(经学生配对检验P&lt；0.01)，并被Calain抑制剂I抑制。为了检测细胞中的Calain和凋亡，我们采用了荧光显微镜下的Boc法和TUNEL法。Boc实验表明，EGF可刺激钙蛋白酶活性。荧光显微镜下的荧光强度被CaPin抑制剂I阻断，TUNEL法阳性细胞数与Boc法一致，证明EGF激活的CaPin以一种依赖于calain的方式诱导细胞凋亡。EGF通过MAP激酶激活信号转导通路，可诱导钙蛋白酶活性和细胞凋亡。EGF可能直接调节角质形成细胞内钙离子浓度，并诱导角质形成细胞中钙蛋白酶的激活，这将是进一步研究的主题，以确定EGF通过哪条信号通路激活角质形成细胞中的钙蛋白酶并诱导细胞凋亡。

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.