Can Yuan

Induction of KLF4 in response to heat stress

Abstract Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger–containing transcription factor with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. However, little is known about the response of KLF4 to heat stress. In this study, Western blot and reverse transcriptase–polymerase chain reaction were performed to determine the changes in KLF4 expression in response to heat stress. The results showed that heat stress up-regulated KLF4 messenger RNA and protein levels in a time-dependent manner in vivo and in 4 cell lines. Moreover, a study with heat shock transcription factor 1 (Hsf1) gene knockout mice indicated that the induction of KLF4 in response to heat stress was mediated by Hsf1. This process occurred rapidly, indicating that KLF4 is an immediate early response gene of heat stress. Next, the roles of KLF4 under heat stress conditions were analyzed for cells overexpressing or deficient in KLF4. The results showed overexpression of KLF4 increased the death rate of C2C12 cells, whereas KLF4 deficiency decreased the injury of C2C12 cells from heat stress conditions, suggesting that KLF4 might play an important role in cell injury induced by heat stress. KLF4 might be an immediate early response gene and could play an important role in cell injury induced by heat stress.

A novel WD-40 repeat protein WDR26 suppresses H2O2-induced cell death in neural cells

WD-40 repeat proteins play important roles in a variety of cellular functions, such as cell growth, proliferation, apoptosis and intracellular signal transduction. We previously identified a novel member of this family, WDR26. To examine the biological function of WDR26, we used hWDR26 plasmids and antisense phosphorothioate oligodeoxynucleotides (asODNs) against WDR26 to examine its role in response to oxidative stress in human SH-SY5Y neuroblastoma cells. Our results showed that H2O2 at 0.5mM substantially induced cell death and significantly up-regulated the WDR26 expression, and WDR26 over-expression in turn strongly suppressed H2O2-induced cell death. Moreover, asODNs markedly inhibited the de novo biosynthesis of WDR26, which contributed to enhanced cell death induced by H2O2. Finally, we found that WDR26 over-expression also down-regulated the transcriptional activity of AP-1 during H2O2-induced SH-SY5Y cell death. Taken together, these results indicated that WDR26 was up-regulated by oxidative stress and played a key role in H2O2-induced SH-SY5Y cell death, which may be mediated by the down-regulation of AP-1 transcriptional activity.

Intracellular Delivery of Recombinant alpha B-crystallin into Neonatal Rat Cardiomyocytes has a Protective Effect on the Cells

In order to deliver alpha B-crystallin (alpha B-C) into cardiomyocytes and study its cellular protection, the full-length cDNA fragment encoding human alpha B-C was cloned into the bacterial expression vector pGEX-MTS containing the base sequence of membrane-translocating sequence (MTS) which mediates intracellular delivery of peptides and expressed as a fusion protein coupled to glutathione S-transferase (GST).After glutathione affinity chromatography and cleaved from GST by factor Xa, the recombinant MTS- alpha B-C was separated from GST and factor Xa by anion exchange chromatography. Recombinant MTS- alpha B-C was characterized by SDS-PAGE and Western immunoblot analysis. The purified MTS- alpha B-C migrated on SDS-PAGE as a single band to an apparent molecular weight (Mr.23kD) that corresponded to total native alpha B-C plus MTS, and was recognized on Western immunoblot by anti-human alpha B-crystallin antibody. MTS- alpha B-C displayed chaperone-like function in an ATP-containing buffer at 37℃ by disaggregating the denatured and aggregated actin induced by hydrogen peroxide (H2O2 )treatment. It was observed under fluorescence microscope that FITC-labeled MTS- alpha B-C had gone into neonatal rat cardiomyocytes by MTS mediation after the cells were incubated with it for 6 hours while FITC-labeled alpha B-C and bovine serum albumin had not gone into the cells. Recombinant MTS- alpha B-C is not cytotoxic, and MTS- alpha B-C-treated cells displayed increased H2O2-tolerance compared with non-treated cells.