| 12-lipoxygenase, is one of lipoxygenases which metabolize AA to 12(S)-hydroxyeicosatetraenoic acids and other bioactive lipids. 12-LOX has been detected in tumor cells and its product, 12(S)-HETE, plays an important role in tumor metastasis and cancer progress. Understanding the regulation of 12-LOX is an important step for prevention of tumor progression. However, the mechanism of 12-LOX regulation is poorly understood. In this study, we hypothesized that there may exist a protein(s) which regulate 12-LOX activity. Using yeast two-hybrid approach to screen human epidermoid carcinoma A431 cDNA library, we identified four intracellular proteins which interact with 12-LOX. They are the cytoplasmic domain of integrin beta4, Keratin K5, Lamin A and phosphoprotein C8FW. We confirmed these interactions in the case of beta4, Lamin A and Keratin K5 by in vitro coimmunoprecipitation assay and confocal immunocolocalization staining. By further characterization of the physical and functional interaction between 12-LOX and beta4, we found that this interaction translocates 12-LOX from cytosol to membrane and consequently upregulates 12LOX enzymatic activity leading to enhanced production of 12(S)-HETE. During the process of translocation, 12-LOX and beta4 are phosphorylated and phosphorylation of 12-LOX is required for the translocation. Furthermore, we showed that beta4 signals via a pathway that prevented apoptosis of A431 either by culture on bacteria plates or with treatment of a 12-LOX inhibitor. 12(S)-HETE may active, by mechanisms not known, PI3 kinase which leads to the phosphorylation of Akt and Bad. This results in decreased apoptosis and provides cells with resistance to apoptosis. Since we showed that AMF stimulates expression of 12-LOX, AMF preventing cells from apoptosis may be also duo to the enhanced production of 12(S)HETE. In addition, beta4 may signal through 12(S)-HETE to stimulate cellular motility. Taken together, the results presented in this dissertation provide a potential mechanism(s) by which 12-LOX activity is regulated in tumor cells and suggest 12-LOX as a potential therapeutic target for interventional cancer therapy. It adds more understanding to the complex role of 12-LOX and integrin beta4 in cancer and other human diseases. |
