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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

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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.

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Acknowledgments

The authors are grateful for the technical assistance of Erle Solheim. The study was financially supported by Grants from the Norwegian South-Eastern Health Authority.

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Authors and Affiliations

  1. Department of Haematology, Oslo University Hospital, Oslo, Norway

    Grethe Skretting, Christiane F. Myklebust, Anders E. Dahm & Per Morten Sandset

  2. Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway

    Grethe Skretting, Christiane F. Myklebust, Anders E. Dahm & Per Morten Sandset

  3. Department of Medical Genetics, Oslo University Hospital, Oslo, Norway

    Nina Iversen

  4. University of Oslo, Oslo, Norway

    Nina Iversen

  5. Institute of Clinical Medicine, University of Oslo, Oslo, Norway

    Per Morten Sandset

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  1. Grethe Skretting
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  2. Nina Iversen
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  3. Christiane F. Myklebust
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  4. Anders E. Dahm
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  5. Per Morten Sandset
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Correspondence to Grethe Skretting.

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Skretting, G., Iversen, N., Myklebust, C.F. et al. 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. Mol Biol Rep 39, 10089–10096 (2012). https://doi.org/10.1007/s11033-012-1882-7

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  • DOI: https://doi.org/10.1007/s11033-012-1882-7

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