Effect of BIS depletion on HSF1-dependent transcriptional activation in A549 non-small cell lung cancer cells

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

ERK-mediated phosphorylation of BIS regulates nuclear translocation of HSF1 under oxidative stress

B-cell lymphoma (BCL)-2-interacting cell death suppressor (BIS) has diverse cellular functions depending on its binding partners. However, little is known about the effects of biochemical modification of BIS on its various activities under oxidative stress conditions. In this study, we showed that H₂O₂ reduced BIS mobility on SDS–polyacrylamide gels in a time-dependent manner via the activation of extracellular signaling-regulated kinase (ERK). The combined results of mass spectroscopy and computational prediction identified Thr285 and Ser289 in BIS as candidate residues for phosphorylation by ERK under oxidative stress conditions. Deletion of these sites resulted in a partial reduction in the H₂O₂-induced mobility shift relative to that of the wild-type BIS protein; overexpression of the deletion mutant sensitized A172 cells to H₂O₂-induced cell death without increasing the level of intracellular reactive oxygen species. Expression of the BIS deletion mutant decreased the level of heat shock protein (HSP) 70 mRNA following H₂O₂ treatment, which was accompanied by impaired nuclear translocation of heat shock transcription factor (HSF) 1. Co-immunoprecipitation assays revealed that the binding of wild-type BIS to HSF1 was decreased by oxidative stress, while the binding of the BIS deletion mutant to HSF1 was not affected. These results indicate that ERK-dependent phosphorylation of BIS has a role in the regulation of nuclear translocation of HSF1 likely through modulation of its interaction affinity with HSF1, which affects HSP70 expression and sensitivity to oxidative stress.

Bis induces growth inhibition and differentiation of HL-60 cells via up-regulation of p27

Bis (Bag-3, CAIR), a Bcl-2-interacting protein, promotes the anti-apoptotic activity of Bcl-2 and increased levels of Bis have been observed in several disease models. The involvement of Bcl-2 and some Bcl-2-binding proteins in differentiation has recently been reported. However, the relevance of Bis to cellular differentiation remains unknown. The findings herein show that Bis expression is up-regulated during the differentiation of HL-60 cells. To investigate the effect of Bis expression on differentiation, we established Bis-overexpressing HL-60 cells (HL-60-bis). HL-60-bis cells have a low nuclear: cytoplasmic ratio and indented nucleus in Wright- Giemsa staining, and an increased expression of CD11b in immunofluorescence study, indicating the promotion of differentiation. The overexpression of Bis also resulted in a retarded cell growth rate, accompanied by the accumulation of HL-60 cells at the G0/G1 phase of the cell cycle, which was sustained during the differentiation process. Western blot analysis revealed that the expression of p27, a representative inducer of cell cycle arrest at the G1 phase, was increased 2.5-fold in HL-60-bis cells compared to HL-60-neo cells. These results suggest that the Bis induced growth inhibition of HL-60 cells promotes G0/G1 phase arrest via up-regulation of p27, which seems to be a prerequisite for differentiation. Further studies will be required to define the exact roles of Bis on cellular differentiation more precisely.

Glycobiology of Selectin / 한국유방암학회지

Selectins, carbohydrate-binding molecules, bind to fucosylated and sialylated glycoprotein ligands, and are found on endothelial cells, leukocytes and platelets. They can be classified into E-, L- and P-selectins, and are involved in trafficking of cells of the innate immune system, T lymphocytes and platelets via binding with specific ligands. An absence of selectins or selectin ligands has serious consequences in mice or humans, leading to recurrent bacterial infections and persistent disease. Selectins are involved in constitutive lymphocyte homing and chronic and acute inflammation processes, including post-ischemic inflammation in muscle, kidney, heart, skin inflammation, atherosclerosis, glomerulonephritis and lupus erythematosus. Selectin-neutralizing monoclonal antibodies, recombinant soluble P-selectin glycoprotein ligand 1 and small-molecule inhibitors of selectins have been tested in clinical trials on patients with multiple trauma, cardiac indications and pediatric asthma, respectively. Anti-selectin antibodies have also been successfully used in preclinical models to deliver imaging contrast agents and therapeutics to sites of inflammation. The contributions of selectins and selectin ligands to signalling deserve further study, which will allow a much more detailed analysis of the contributions of selectins in models of inflammation, haemostasis, haematopoiesis, wound healing, atherogenesis, and tumor metastasis.