| Summary: | 博士 === 國立中央大學 === 生命科學系 === 102 === Endothelin (ET)-1, suppressor of cytokine signaling (SOCS)-3 and epigallocatechin gallate (EGCG) are important to regulate the lipid metabolism and insulin resistance, respectively. However, no clear evidence is showed the relationship among ET-1, SOCS-3 and EGCG. This dissertation was designed to understand the effect of ET-1 on modulating the SOCS-3 gene and further investigate whether EGCG regulated the effect of ET-1 on SOCS-3 gene.
Chapter One indicated that ET-1 upregulated the expression of SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, and SOCS-6 mRNAs, but not SOCS-7 or cytokine-inducible SH2-containing protein (CIS)-1 mRNAs. The ET-1 stimulation of SOCS-3 mRNA expression required new RNA synthesis and was cell-type specific. The stimulatory effects of ET-1 on SOCS-3 mRNA and protein expression were mediated through the ERK, JNK, PI3K, and JAK2 pathways.
Chapter Two showed that EGCG suppressed the ET-1-induced expression of the SOCS-3 gene in 3T3-L1 adipocytes through the 67LR and AMPK pathways. EGCG also suppressed the ET-1-stimulated expression of SOCS-1, -2, -4, -5 and -6 mRNAs, but not SOCS-7 or CIS-1 mRNAs. EGCG inhibited the ET-1-increased phosphorylation of different ET-1 signaling proteins, such as ERKs, p38, JNK, cJUN, AKT, JAK, and, to a lesser extent, STAT-3 proteins.
Chapter Three indicated that 67LR gene was isolated and sequenced with 885 bp and 295 amino acid (aa) from murine primary and secondary adipocytes and it had the 96% and 89% homology of nucleotide sequence to rat and human. The 67LR expression depended on tissue types and growth status. Treatment with 67LR antiserum blocked the inhibitory effect of EGCG on cell number in 3T3-L1 preadipocytes, suggesting the 67LR-dependent effect.
Chapter Four showed that different plasmids for recombinant full-length (67LR1-295) and truncated forms (67LR1-200, 67LR1-150, 67LR1-100 and 67LR1-55) of human and mouse 67LR were constructed with a Flag tag and stably cloned in KB oral cancer cells and MCF-7 breast cancer cells were established. Growth experiments indicated mitogenic effect of 67LR on cancer cells varies with cell types and different domains of the 67LR protein. Different forms of 67LR stably cloned MCF-7 and KB cells treated with EGCG showed that amino acid residues of the 67LR from 151-200 are important for modulating the antimitogenic effects of EGCG on MCF-7 and KB cancer cells.
Chapter Five was successful to generate one heterozygous 67LR gene (also called Rpsa-/flox alleles), the mice with Rpsa-/- alleles would be needed for a further study.
We conclude that ET-1 acts particular types of SOCS family members through the ETAR, ERK, JNK, JAK and PI3K but not ETBR pathways. The anti-ET-1 signaling effect of EGCG is mediated by 67LR and AMPK pathways. As the 67LR was discovered as an EGCG receptor and as the SOCS proteins were reported as an insulin signaling inhibitor, results of this dissertation could help explain the mechanism of how EGCG and ET-1 interacts on adipocyte functions and insulin signaling.
|
|---|
