Study of the Associated Proteins of STAT3 and Characterization of their Functions: Roles of GRIM-19 and PIN1 in the Regulation of STAT3 Activity

Ph.DDOCTOR OF PHILOSOPH

STAT3-Inducible Mouse ESCs: A Model to Study the Role of STAT3 in ESC Maintenance and Lineage Differentiation

Studies have demonstrated that STAT3 is essential in maintaining self-renewal of embryonic stem cells (ESCs) and modulates ESC differentiation. However, there is still lack of direct evidence on STAT3 functions in ESCs and embryogenesis because constitutive STAT3 knockout (KO) mouse is embryonic lethal at E6.5–E7.5, prior to potential functional role in early development can be assessed. Therefore, in this study, two inducible STAT3 ESC lines were established, including the STAT3 knockout (InSTAT3 KO) and pSTAT3 overexpressed (InSTAT3 CA) using Tet-on inducible system in which STAT3 expression can be strictly controlled by doxycycline (Dox) stimulation. Through genotyping, deletion of STAT3 alleles was detected in InSTAT3 KO ESCs following 24 hours Dox stimulation. Western blot also showed that pSTAT3 and STAT3 protein levels were significantly reduced in InSTAT3 KO ESCs while dominantly elevated in InSTAT3 CA ECSs upon Dox stimulation. Likewise, it was found that STAT3-null ESCs would affect the differentiation of ESCs into mesoderm and cardiac lineage. Taken together, the findings of this study indicated that InSTAT3 KO and InSTAT3 CA ESCs could provide a new tool to clarify the direct targets of STAT3 and its role in ESC maintenance, which will facilitate the elaboration of the mechanisms whereby STAT3 maintains ESC pluripotency and regulates ESC differentiation during mammalian embryogenesis

GRIM-19, a Cell Death Regulatory Protein, Is Essential for Assembly and Function of Mitochondrial Complex I

Mitochondria play essential roles in cellular energy production via the oxidative phosphorylation system (OXPHOS) consisting of five multiprotein complexes and also in the initiation of apoptosis. NADH:ubiquinone oxidoreductase (complex I) is the largest complex that catalyzes the first step of electron transfer in the OXPHOS system. GRIM-19 was originally identified as a nuclear protein with apoptotic nature in interferon (IFN)- and all-trans-retinoic acid (RA)-induced tumor cells. To reveal its biological role, we generated mice deficient in GRIM-19 by gene targeting. Homologous deletion of GRIM-19 causes embryonic lethality at embryonic day 9.5. GRIM-19(−/−) blastocysts show retarded growth in vitro and, strikingly, display abnormal mitochondrial structure, morphology, and cellular distribution. We reexamined the cellular localization of GRIM-19 in various cell types and found its primary localization in the mitochondria. Furthermore, GRIM-19 is detected in the native form of mitochondrial complex I. Finally, we show that elimination of GRIM-19 destroys the assembly and electron transfer activity of complex I and also influences the other complexes in the mitochondrial respiratory chain. Our result demonstrates that GRIM-19, a gene product with a specific role in IFN-RA-induced cell death, is a functional component of mitochondrial complex I and is essential for early embryonic development