Studies on low rigidity of collagen gel-induced apoptosis and autophagy in epithelial cells

• Main Authors: Yao-Hsien Wang, 王耀賢
• Other Authors: Ming-Jer Tang
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/89126761986697020225

博士 === 國立成功大學 === 基礎醫學研究所 === 95 === In this thesis, I established that collagen gel, but not collagen gel-coating, induced apoptosis exclusively in epithelial cell lines, which indicated that low substratum rigidity might trigger cell apoptosis. Alternation of collagen gel structure by age effect or sonication could decrease or elevate apoptosis ratio, respectively. I found that collagen-gel-induced apoptosis was inversely correlated with substratum rigidity. Low-substratum-rigidity collagen-gel-induced apoptosis was neither prevented by overexpression of Bcl-2 nor preceded by mitochondrial release of cytochrome c. Low substratum rigidity activated c-Jun N-terminal kinase (JNK), but it also rapidly downregulated c-Jun and triggered persistent aberrant expression of c-Fos. Either reduction of c-Jun expression or overexpression of c-Fos has been shown to induce apoptosis in several epithelial cells. Inhibition of low-substratum-rigidity-induced JNK activation prevented aberrant c-Fos expression, but only partially blocked low-substratum-rigidity-induced apoptosis. Taking these results together, I conclude that low-substratum-rigidity collagen gel induced apoptosis in epithelial cells, and that deregulation of AP-1 proteins mediated that apoptosis, at least in part. The second project was to examine the mechanism whereby collagen gel induced a delay apoptosis in epithelial cells. Low rigidity of collagen gel induced apoptosis in epithelial cells within 24 h, whereas it induced an increase in intracellular Ca++ ([Ca++]i) and endoplasmic reticulum Ca++ overloading within 1 h, which lead to cell membrane flipping as assessed by positive Annexin V staining. In addition, low substratum rigidity triggered deregulation of AP-1 protein family, which eventually resulted in apoptosis within 24 h, as reflected by the presence of DNA and nuclear fragmentation. In order to understand the sequential biological events between membrane flipping and nuclear fragmentation as triggered by low substratum rigidity, I would like to know whether the formation of autophagy is involved in this process. I found that low substratum rigidity of collagen gel induced the formation of autophagy in normal epithelial but not transformed cells within 8 h. Inhibition of autophagy formation augmented nuclear fragmentation without alteration of membrane flipping at the early phase. Reduction of [Ca++]i by calcium chelators or channel blockers prevented low rigidity-induced formation of autophagy. In contrast, treatment of Ca++ ionophore induced formation of autophagy, suggesting that low substratum rigidity-induced intracellular Ca++ overload leads to the formation of autophagy. Moreover, low rigidity of collagen gel induced activation of m-calpain. However, activation of m-calpain was not involved in the formation of autophagy or degradation of c-Jun. Taken together, I conclude that low rigidity induces an increase in [Ca++]i and triggers formation of autophagy which contributes to delay low rigidity-induced apoptosis mediated by c-Jun degradation.