The transcription complex Prep1-Pbx1 regulates the gene expression of tyrosine phosphatases and impairs insulin action in liver cells
Iovino, Salvatore (2009) The transcription complex Prep1-Pbx1 regulates the gene expression of tyrosine phosphatases and impairs insulin action in liver cells. [Tesi di dottorato] (Inedito)
Full text disponibile come:
Prep1 is an homeodomain transcription factor belonging to the MEINOX subfamily of the TALE (three amino acid loop extension) proteins. Prep1 forms DNA-independent dimeric complexes with all isoforms of the Pbx homeodomain transcription factor, enhancing target specificity and regulatory function. Recently, we have shown that Prep1 deficiency in mice induces protection from diabetes and increased insulin sensitivity in muscle tissue through a mechanism which involves increased protein and mRNA levels of the glucose transporter (GLUT)-4 and the PPAR gamma coactivator-1 alpha (PGC-1. Since PGC-1promotes the gluconeogenesis in hepatic tissue, I have studied the role of Prep1 in regulating insulin signaling in liver of Prep1 hypomorphic (Prep1i/i, Prep1i/+) mice, which expressed respectively only 5-7% and 55-57% of protein. Despite the results obtained in muscle tissue, Prep1i/i and Prep1i/+ mice did not show changes in PGC-1protein and mRNA levels, but surprisingly they have an improved insulin-stimulated phosphorylation of IR and IRS-1/2. This is paralleled by an increase of glycogen content and a reduction of Glucose-6-phosphatase and PEPCK expression. Western blot analysis and qRT-PCR experiments displayed a gene-dosage dependent reduction of protein and mRNA levels of SHP1 and SYP tyrosine phosphatases in liver extracts of Prep1i/i, Prep1i/+ mice. In parallel, the overexpression of Prep1 and Pbx1 in and HepG2 (Human Hepatoma cell line) cells induced insulin resistance by increasing the protein content and mRNA expression of SHP1 and SYP phosphatases, which were paralleled by an inhibition of insulin-stimulated phosphorylation of IR, IRS-1/2 and glycogen accumulation. Interestingly, the overexpression of an inactive form of Prep1 (Prep1HR1), lacking of the interaction site between Prep1 and Pbx1, did not impair SHP1 and SYP expression and insulin-signaling in HepG2 cells. Moreover, in Prep1 overexpressing cells, antisense silencing of SHP1 but not that of SYP rescued insulin-dependent IR phosphorylation and glycogen accumulation. Finally, ChIP and Re-Chip experiments pointed out that the dimeric complex Prep1-Pbx1 bound specific sequences upstream the ATG codon of SYP and SHP1 genes (-625bp and -2113bp respectively) suggesting a direct gene regulation. Luciferase assays confirmed that the regions upstream SYP and SHP1 genes, were functionally activated by Prep1 and Pbx1 overexpression. Thus, the dimeric complex Prep1-Pbx1 directly regulates the gene expression of SYP and SHP1 tyrosine phosphatases by promoting insulin-resistance in liver cells.
Solo per gli Amministratori dell'archivio: edita il record