Intracellular Redox Compartments in Hypoxia-Signalling and Associated Diseases

Project Leader: Prof. Thomas Kietzmann, M.D., Ph.D.
Faculty of Biochemistry and Molecular Medicine,
University of Oulu

Background and Significance

Adaptation to changes in ambient O2 tension is essential for adequate energy supply in humans and all other aerobic living organisms. Disturbances in O2 and nutrient supply have profound effects not only on cellular but also whole body function and may contribute to the development of various diseases such as cancer, diabetes mellitus, metabolic syndrome, artherosclerosis and thrombosis. The α-subunits of HIFs play key roles in cellular responses to hypoxia and the pathogenesis of these diseases. Major regulation of HIFs occurs at the level of protein stability; the actions of HIF proline hydroxylases allow subsequent recruitment of the pVHL ubiquitin ligase complex, which enables proteasomal degradation of HIFs. Moreover, compelling evidence has been obtained for the existence of other, non-pVHL-mediated HIF-1α degradation mechanisms. One example is our finding showing degradation of HIF-1α via a new pathway involving Fbw7 and USP28. In addition, a number of reports including our own have shown that HIFs also respond to growth and coagulation factors, hormones, cytokines and stress factors under non-hypoxic conditions by using ROS as mediators. Although these data show that ROS levels have an impact on HIF-1α levels, there are diverse opinions about the enzymes and cellular compartments participating in ROS generation. Thus, different cellular compartments such as the endoplasmic reticulum, the Golgi complex and the mitochondria may have different roles in redox signalling.

Recent Progress

Our research in the past has shown that HIFs respond to growth and coagulation factors (PDGF, IGF-1, thrombin), hormones (insulin) and cytokines (TNFα) in normoxic conditions by using ROS as mediators. Thereby, different cellular compartments and therein localized enzymes such as NOX4, an ER-based member of the NADPH oxidases, and a ROS-generating Fenton reaction at the ER can modulate HIF-1α levels. At the same time hypoxia and ROS can also activate the HIF-1α promoter by involving phosphatidylinositol 3-kinase and a functional NFkappaB site in the hif-1α gene promoter.

Moreover, we were the first to discover the molecular mechanisms by which hypoxia activates transcription of plasminogen activator inhibitor-1 (PAI-1), which is a breast cancer marker as well as being associated with obesity. Resveratrol, a constituent of grapes and berries, was proposed to improve obesity-related health problems, and by using SGBS adipocytes and a model of human adipose tissue inflammation we showed that treatment of SGBS adipocytes with resveratrol reduced PAI-1 gene expression. Although signalling via PI3K, Sirt1, AMPK, ROS and Nrf2 appeared to play a significant role in the modulation of PAI-1 gene expression under non-inflammatory conditions, these signalling components were not involved in mediating the effects of resveratrol on PAI-1 production under inflammatory conditions. Instead, we demonstrated that the effects of resveratrol on PAI-1 induction under inflammatory conditions were mediated via inhibition of the NFkB pathway. Thus, resveratrol can act as an NFkB inhibitor in adipocytes and the subsequently reduced PAI-1 expression in inflamed adipose tissue might provide new insight as regards novel options in the treatment of obesity.

In addition to the established finding that PAI-1 serves as a breast cancer marker indicating a bad prognosis, we found that the epidermal growth factor (EGF) receptor Her1 adaptor protein CIN85 is expressed at very high levels in samples from patients with invasive breast adenocarcinomas. We also found that CIN85 can increase the levels of HIF-1α as a result of HIF-1α protein stabilization. In addition, we showed that HIF-1 signalling is important for the insulin-(protein kinase B/Akt)-dependent induction of PAI-1. In line with insulin-dependent HIF α-subunit regulation, we showed that the PKB/Akt target GSK-3 initiates VHL-independent HIF-1α degradation. In line with these observations, we found that GSK-3 induced phosphorylation and recruitment of ubiquitin ligase and the tumour suppressor F-box and WD protein Fbw7. Further, GSK-3β- and Fbw7-dependent HIF-1α degradation can be antagonized by ubiquitin-specific protease-28 (USP28). Altogether, we identified a new pathway which can influence HIF-1α-dependent processes.

Future Goals

We defined three major goals: i) to determine the role of compartment-specific ROS generation in HIF signalling, ii) to understand the mechanism by which CIN85 induces HIF-1a and iii) to identify the role of USP28 in cancerogenesis.

Our first goal is to investigate how compartment-specific concentration changes of ROS modulate the activity of the HIFa family and expression of their target genes. In particular, we will address the question of whether or not disturbances of ROS balance in mitochondria, peroxisomes or in the ER have an impact on HIFa expression and degradation in vivo and in vitro. To investigate the impact of the different compartments in vivo we will first use our mice in which the mitochondrial antioxidative enzyme MnSOD (SOD2) has been knocked out specifically in hepatocytes. These mice can be placed in special hypoxia chambers and induction of HIFa proteins can be investigated. These studies will be accompanied by cell culture experiments with either immortalized hepatocytes or mouse embryonic fibroblasts derived from knockout mice. To extend these analyses we also aim to investigate HIFa levels upon knockdown of other components of various compartments (e.g. Gpx, Mpv17, catalase, ERp72). In addition, it is necessary to monitor ROS changes with respect to different cellular compartments, something which has not yet been done. We will realize this by combining two-photon confocal laser scanning microscopy together with redox-sensitive green fluorescent proteins that are targeted specifically to different cellular compartments.

As regards our second goal we will continue to unravel the mechanisms by which CIN85 interferes with HIF degradation and investigate whether or not it interferes with PHD function. So far, it is unknown how these molecules interact and which domains would be involved.

Work on the third goal will include systematic screening for further USP28-interacting proteins with the help of tandem affinity chromatography and mass spectrometry. This system should also enable us to identify proteins specifically interacting with CIN85. In these analyses we will greatly benefit from the expertise and equipment available in the Biocenter Oulu Proteomics and Protein Analysis core facility. The impact of the identified USP28 interactors on HIFs, PAI-1 expression, insulin-regulated metabolism as well as angiogenesis will then be studied.

Novel outcomes can be expected by combining the work of our group with expertise on mitochondria, peroxisomes and ER available in Oulu.

Publications 2011-

Fratz S, Fineman JR, Görlach A, Sharma S, Oishi P, Schreiber C, Kietzmann T, Adatia I, Hess J, Black SM. Early determinants of pulmonary vascular remodeling in animal models of complex congenital heart disease. Circulation 123:916-923, 2011.

Flügel D, Görlach A, Kietzmann T. GSK-3beta regulates cell growth, migration, and angiogenesis via Fbw7 and USP28-dependent degradation of HIF-1alpha. Blood 119:1292-1301, 2012.

Samoylenko A, Vynnytska-Myronovska B, Byts N, Kozlova N, Basaraba O, Pasichnyk G, Palyvoda K, Bobak Y, Barska M, Mayevska O, Rzhepetsky Y, Shuvayeva H, Lyzogubov V, Usenko V, Savran V, Volodko N, Buchman V, Kietzmann T, Drobot L. Increased levels of the HER1 adaptor protein Rukl/CIN85 contribute to breast cancer malignancy.  Carcinogenesis 33:1976-1984, 2012.

Samoylenko A, Hossain JA, Mennerich D, Kellokumpu S, Hiltunen JK, Kietzmann T. Nutritional countermeasures targeting reactive oxygen species in cancer: from mechanisms to biomarkers and clinical evidence. Antiox Redox Signal 19:2157-2196, 2013.

Zagotta I, Dimova EY, Funcke JB, Wabitsch M, Kietzmann T, Fischer-Posovszky P. Resveratrol suppresses PAI-1 gene expression in a human in vitro model of inflamed adipose tissue. Oxid Med Cell Longe 2013, 793525, 2013.

Chi FT, Jensen JK, Kozlova N, Samoylenko A, Kietzmann T. PAI-1 modulates cell migration in a LRP1-dependent manner via β-catenin and ERK1/2. Thromb Haemost. in press.

Horbach T, Chi TF, Götz C, Sharma S, Juffer AH, Dimova EY, Kietzmann T. GSK3β-Dependent phosphorylation alters DNA binding, transactivity and half-life of the transcription factor USF2. PLoS One 9(9):e107914, 2014.

Horbach T, Goetz C, Kietzmann T, Dimova, EY. Protein kinases as switches for the function of upstream stimulatory factors (USFs) in cancer? Front Pharm. in press.

Kinnunen M, Karmenyanb, Särkeläa A, Dimova EY, Kietzmann T. Low-intensity light detection methods for selected biophotonic applications. Proc. SPIE 9421, Eighth International Conference on Advanced Optical Materials and Devices (AOMD-8), doi:10.1117/12.2084915; 2014

Konzack A, Kietzmann T. Manganese superoxide dismutase in carcinogenesis: friend or foe? Biochem Soc Trans. 42(4): 1012-1016, 2014.

Kozlova N, Samoylenko A, Drobot L, Kietzmann T. Urokinase is a negative modulator of Egf-dependent proliferation and motility in the two breast cancer cell lines MCF-7 and MDA-MB-231. Mol Cancerogenesis, in press.

Lupp S, Khadouma S, Horbach T, Dimova EY, Bohrer AM, Kietzmann T, Montenarh M, Götz C. The upstream stimulatory factor USF1 is regulated by protein kinase CK2 phosphorylation. Cell Signal 26(12):2809-2817, 2014.

Research Group Members

Project Leader:
Thomas Kietzmann, M.D., Ph.D., Professor (University of Oulu)

Senior and Post-doctoral Investigators:
Daniela Mennerich, Ph.D. (Biocenter Oulu)

Ph.D. Students:
Nina Kozlova, M.Sc. (Biocenter Oulu)
Kati Richter, M.Sc. (University of Oulu)
Anja Konzack, M.Sc. (University of Oulu)
Franklin Tabughang Chi, M.Sc. (Sigrid Jusélius Foundation)

Laboratory Technicians:
Maire Jarva (University of Oulu)

Main source of salary in brackets.

Foreign Scientists, 7

National and International Activities

Group Members Who Spent More Than Two Weeks in Foreign Laboratories During 2014

Elitsa Dimova, Erasmus University Rotterdam, The Netherlands

EU Projects (present and progress)

EU-ROS; COST Action: BM1203; MC member substitute

Last updated: 23.3.2016