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Putative Role of Nuclear Factor-Kappa B But Not Hypoxia-Inducible Factor-1α in Hypoxia-Dependent Regulation of Oxidative Stress in Hematopoietic Stem and Progenitor Cells
Linköping University, Department of Clinical and Experimental Medicine, Division of Hematopoiesis and Developmental Biology. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Hematopoiesis and Developmental Biology. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Hematopoiesis and Developmental Biology. Linköping University, Faculty of Medicine and Health Sciences.
Lund Stem Cell Center, Lund University, Lund, Sweden.
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2019 (English)In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 31, no 3, p. 211-226Article in journal (Refereed) Published
Abstract [en]

Aims: Adaptation to low oxygen of hematopoietic stem cells (HSCs) in the bone marrow has been demonstrated to depend on the activation of hypoxia-inducible factor (HIF)-1α as well as the limited production of reactive oxygen species (ROS). In this study, we aimed at determining whether HIF-1α is involved in protecting HSCs from ROS.

Results: Oxidative stress was induced by DL-buthionine-(S,R)-sulfoximine (BSO)-treatment, which increases the mitochondrial ROS level. Hypoxia rescued Lineage-Sca-1+c-kit+ (LSK) cells from BSO-induced apoptosis, whereas cells succumbed to apoptosis in normoxia. Apoptosis in normoxia was inhibited with the antioxidant N-acetyl-L-cysteine or by overexpression of anti-apoptotic BCL-2. Moreover, stabilized expression of oxygen-insensitive HIFs could not protect LSK cells from oxidative stress-induced apoptosis at normoxia, neither could short hairpin RNA to Hif-1α inhibit the protective effects by hypoxia in LSK cells. Likewise, BSO treatment of LSK cells from Hif-1α knockout mice did not suppress the effects seen in hypoxia. Microarray analysis identified the nuclear factor-kappa B (NF-κB) pathway as a pathway induced by hypoxia. By using NF-κB lentiviral construct and DNA-binding assay, we found increased NF-κB activity in cells cultured in hypoxia compared with normoxia. Using an inhibitor against NF-κB activation, we could confirm the involvement of NF-κB signaling as BSO-mediated cell death was significantly increased in hypoxia after adding the inhibitor.

Innovation: HIF-1α is not involved in protecting HSCs and progenitors to elevated levels of ROS on glutathione depletion during hypoxic conditions.

Conclusion: The study proposes a putative role of NF-κB signaling as a hypoxia-induced regulator in early hematopoietic cells.

Place, publisher, year, edition, pages
Mary Ann Liebert, 2019. Vol. 31, no 3, p. 211-226
Keywords [en]
hematopoiesis, hypoxia, oxidative stress, glutathione, mitochondria, NF-κB
National Category
Cell and Molecular Biology Cell Biology Immunology
Identifiers
URN: urn:nbn:se:liu:diva-156661DOI: 10.1089/ars.2018.7551ISI: 000464553400001PubMedID: 30827134OAI: oai:DiVA.org:liu-156661DiVA, id: diva2:1313739
Note

Funding agencies: Swedish Research Council; Swedish Cancer Foundation; Swedish Childrens Cancer Foundation; County Council of Ostergotland

Available from: 2019-05-06 Created: 2019-05-06 Last updated: 2019-10-17Bibliographically approved
In thesis
1. Hypoxia inducible factor 1 alpha: dependent and independent regulation of hematopoietic stem cells and leukemia
Open this publication in new window or tab >>Hypoxia inducible factor 1 alpha: dependent and independent regulation of hematopoietic stem cells and leukemia
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis has studied the role of low oxygen levels, or hypoxia, in hematopoietic stem cells (HSCs) and how, at the molecular level, it regulates stem cell maintenance and protects against oxidative stress induced by reactive oxygen species (ROS). HSCs reside within the bone marrow in specific niches created by a unique vascularized environment, which is suggested to be hypoxic and crucial for HSCs by maintaining a quiescent state of cell cycle and by redirecting metabolism away from the mitochondria to glycolysis. The niches are also believed to limit the production of ROS, which could damage DNA and disrupt the stem cell features. The hypoxia-responsive protein hypoxia-inducible factor 1 alpha (HIF-1α) is a major regulator of the hypoxic cell response in HSCs as well as in leukemic stem cells. Both these cells are thought to reside in the bone marrow where they are protected from stress and chemotherapy by niche cells and hypoxia.

The thesis demonstrates that pyruvate dehydrogenase kinase 1 regulates a metabolic shift to glycolysis, and maintains the engraftment potential of both HSCs and multipotent progenitors upon transplantation. Furthermore, we wanted to determine whether HIF-1α or other signaling pathways are involved in protecting HSCs from ROS-induced cell death. Overexpression, silencing or a knockout mouse model of Hif-1α could not identify HIF-1α as important for protecting HSCs from oxidative stress-induced cell death through inhibition of synthesis of the antioxidant glutathione. Gene expression analysis instead identified the transcription factor nuclear factor kappa B (NF-κB) as induced by hypoxia. By studying NF- κB signaling we found increased NF-κB activity in cells cultured in hypoxia compared to normoxia. Suppression of inhibitor of kappa B indicated a putative role of NF-κB signaling in hypoxia-induced protection against oxidative stress. The findings show that hypoxia-induced protection to elevated levels of ROS upon glutathione depletion seems to be attributed to activation of the NF-κB signaling pathway independently of HIF-1α.

To address the question whether hypoxic in vitro cultures support maintenance and promote HSC expansion we performed a limited dilution-transplantation assay. Our data indicate that hypoxic cultures maintain more long-term-reconstituting HSCs than normoxia, but this could not be confirmed statistically. Finally, we wanted to study the mechanisms by which hypoxia protect against chemotherapy. We could demonstrate that hypoxic culture protects leukemic cell lines against apoptosis induced by chemotherapy or inhibitors used for treatment of leukemia. This multidrug resistance seems to be mediated by ATP-binding cassette transporter genes, which are upregulated by hypoxia and whose inhibition has been shown to increase chemosensitivity. In addition, HIF-1α was upregulated in the leukemic cell lines in hypoxia and its inhibition increased the sensitivity to chemotherapy, indicating a role in inducing chemotherapy resistance.

Conclusively, the results presented in this thesis stress the importance of hypoxia in regulating metabolism, oxidative-stress response and maintenance of both HSCs as well as leukemic cells, especially through the critical transcription factors HIF-1α and NF-κB and their target genes.  

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. p. 71
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1643
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-152129 (URN)10.3384/diss.diva-152129 (DOI)9789176852101 (ISBN)
Public defence
2018-12-14, Hasselquistsalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2018-10-29 Created: 2018-10-18 Last updated: 2019-09-30Bibliographically approved

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