| Summary: | 博士 === 國立陽明大學 === 微生物及免疫學研究所 === 104 === Porphyrins are a family of heterocyclic organic compounds which compose of four pyrrole rings interconnected via methane bridges. Heme is the most abundant type of porphyins in mammalians which has been used to generate hemoproteins that involves in multiple cellular functions such as oxygen sensing, electron transfer, xenobiotic metabolism, and so on. However, excess of free heme or its precursor, protoporphyrin IX (PPIX) can lead to increase in the level of reactive oxygen species (ROS) that resulted in tissue damage. Therefore, controlling intracellular levels of heme and PPIX is important for cell and tissue homeostasis. Previously, ABCG2 has been identified as a heme/PPIX exporter which located in cytoplasmic and mitochondrial membranes. However, the role of ABCG2 in regulating intracellular heme levels in adult tissue needed to be further elucidated. Therefore, in the current thesis work, I investigated the effects of the disturbance of cellular heme levels in ABCG2-deficient status. Especially, we used ABCG2-knockout mice to study the role of ABCG2 in regulating intracellular heme levels in normal and injured skin and liver.
In the first part of this thesis work, I investigated the role of ABCG2 in cutaneous wound healing. Full-thickness skin wounds were created in ABCG2 knockout (ABCG2-KO) and wild-type mice. The healing process was then analyzed and revealed that ABCG2 deficiency in skin results in delays in wound closure and impairments in re-epithelialization, as evidenced by reductions in both suprabasal differentiation and in p63-expressing keratinocytes migrating from transitional epidermis to epithelial tongues. To further determine if ABCG2 deficiency affects the potency of epidermal stem/progenitor cells (EPCs), transplantation studies were carried out, which demonstrated that ABCG2-KO EPCs display higher levels of H2AX and lose the capacity to differentiate into suprabasal keratinocytes. A competitive repopulation assay confirmed that ABCG2 expression is critical for the proper expansion and differentiation of EPCs in cutaneous wounds. Since EPCs are known to contribute to the healing of larger wounds, the findings imply a functional role for ABCG2 in the expansion and differentiation of p63-expressing EPCs.
In the second part of this thesis work, we initially identified homeostasis of intracellular levels of protoporphyrin IX in hepatocytes is essential to maintain mitochondrial dynamics and functions. The increased levels of cytoplasmic and mitochondrial PPIX in ABCG2-deficient hepatocytes would lead to ROS accumulation and to promotion of mitochondria fission mediated by activated p53. To further investigate how excessive levels of porphyrins can affect the potency of liver stem/progenitor cells under porphyria-induced liver injury, I setup 3,5-diethoxycarbonyl-1,4-dihydrochollidine (DDC) treated mice models and found N-methyl-PPIX is produced accompanied by induction of Sox-9 expression in peri-portal hepatocytes. The further experiment confirmed N-methyl-PPIX actually triggered Sox9 expression in primary hepatocytes. In contrast, ABCG2 deficiency could restrict N-methyl-PPIX accumulation and Sox9 induction. Collectively, the findings indicate ABCG2 is required for DDC-induced porphyria in mice and N-methyl-PPIX possibly plays a role in triggering Sox9-mediated hepatic response in the injured liver.
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