title: Overexpression of tissue factor pathway inhibitor in CHO-K1 cells results in increased activation of NF-κB and apoptosis mediated by a caspase-3 independent pathway.
abstract: There is now circumstantial evidence that tissue factor pathway inhibitor (TFPI) is not only a major anticoagulant, but also has proapoptotic properties. The current study was designed to address the role of TFPI on signalling pathways and apoptosis. The non-TFPI expressing cell line CHO-K1 was stably transfected with pcDNA3.1/V5-His-TOPO-TFPI and control cells were established by transfecting the CHO-K1 cells with pcDNA3.1/V5-His-TOPO. Sodium butyrate (NaBut) has been shown to induce the expression of recombinant proteins. Here we have used NaBut to increase the expression of TFPI as assessed by qRT-PCR and ELISA. Compared to the control cells, TFPI induced apoptosis in a concentration dependent manner as measured by a cell death detection assay. Independent of caspase-3 activation an increased cleavage of PARP was detected in the TFPI expressing cells. This was accompanied by downregulation of Bcl-XL, elevated levels of Bax, and increased translocation of the apoptosis initiating factor. Increased DNA binding activity of NF-κB was revealed by electrophoretic mobility shift assay when the TFPI level was elevated by NaBut together with an increased translocation of the NF-κB subunit p65. The results indicate that TFPI affected the apoptotic activity through a process independent of caspase-3, and was also able to increase the activation of the NF- κB pathway.
authors.affiliation: Department of Haematology, Oslo University Hospital, Oslo, Norway. grethe.skretting@medisin.uio.no
authors.forename: Grethe
authors.initials: G
authors.lastname: Skretting
authors.forename: Nina
authors.initials: N
authors.lastname: Iversen
authors.forename: Christiane F
authors.initials: CF
authors.lastname: Myklebust
authors.forename: Anders E
authors.initials: AE
authors.lastname: Dahm
authors.forename: Per Morten
authors.initials: PM
authors.lastname: Sandset
mesh_terms: D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D020148:Butyric Acid / Q000494:pharmacology; D016466:CHO Cells; D053148:Caspase 3 / Q000378:metabolism*; D006224:Cricetinae; D015870:Gene Expression; D006801:Humans; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D051017:Apoptosis Regulatory Proteins; D008074:Lipoproteins; D016328:NF-kappa B; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D020148:Butyric Acid; D019869:Phosphatidylinositol 3-Kinases; D053148:Caspase 3