Skip to main content

Advertisement

SpringerLink
Log in

Impairment of hypoxia-induced HIF-1α signaling in keratinocytes and fibroblasts by sulfur mustard is counteracted by a selective PHD-2 inhibitor

  • Molecular Toxicology
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

Abstract

Skin exposure to sulfur mustard (SM) provokes long-term complications in wound healing. Similar to chronic wounds, SM-induced skin lesions are associated with low levels of oxygen in the wound tissue. Normally, skin cells respond to hypoxia by stabilization of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α modulates expression of genes including VEGFA, BNIP3, and MMP2 that control processes such as angiogenesis, growth, and extracellular proteolysis essential for proper wound healing. The results of our studies revealed that exposure of primary normal human epidermal keratinocytes (NHEK) and primary normal human dermal fibroblasts (NHDF) to SM significantly impaired hypoxia-induced HIF-1α stabilization and target gene expression in these cells. Addition of a selective inhibitor of the oxygen-sensitive prolyl hydroxylase domain-containing protein 2 (PHD-2), IOX2, fully recovered HIF-1α stability, nuclear translocation, and target gene expression in NHEK and NHDF. Moreover, functional studies using a scratch wound assay demonstrated that the application of IOX2 efficiently counteracted SM-mediated deficiencies in monolayer regeneration under hypoxic conditions in NHEK and NHDF. Our findings describe a pathomechanism by which SM negatively affects hypoxia-stimulated HIF-1α signaling in keratinocytes and fibroblasts and thus possibly contributes to delayed wound healing in SM-injured patients that could be treated with PHD-2 inhibitors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

BNIP3:

BCL2/adenovirus E1B 19-kDa protein-interacting protein 3

HIF-1α:

Hypoxia-inducible factor 1 alpha

MMP-2:

Matrix metalloproteinase 2

MMP-9:

Matrix metalloproteinase 9

NHDF:

Normal human dermal fibroblasts

NHEK:

Normal human epidermal keratinocytes

PHD-2:

Prolyl hydroxylase domain-containing protein 2

VEGF-A:

Vascular endothelial growth factor a

SM:

Sulfur mustard

References

  • Balali-Mood M, Hefazi M, Mahmoudi M et al (2005) Long-term complications of sulphur mustard poisoning in severely intoxicated Iranian veterans. Fundam Clin Pharmacol 19(6):713–721

    Article  CAS  PubMed  Google Scholar 

  • Bellot G, Garcia-Medina R, Gounon P et al (2009) Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol Cell Biol 29(10):2570–2581

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Benita Y, Kikuchi H, Smith AD, Zhang MQ, Chung DC, Xavier RJ (2009) An integrative genomics approach identifies hypoxia inducible factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic Acids Res 37(14):4587–4602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bruick RK (2000) Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc Natl Acad Sci USA 97(16):9082–9087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Buth H, Wolters B, Hartwig B et al (2004) HaCaT keratinocytes secrete lysosomal cysteine proteinases during migration. Eur J Cell Biol 83(11–12):781–795

    Article  PubMed  Google Scholar 

  • Catrina SB, Okamoto K, Pereira T, Brismar K, Poellinger L (2004) Hyperglycemia regulates hypoxia-inducible factor-1alpha protein stability and function. Diabetes 53(12):3226–3232

    Article  CAS  PubMed  Google Scholar 

  • Chowdhury R, Candela-Lena JI, Chan MC et al (2013) Selective small molecule probes for the hypoxia inducible factor (HIF) prolyl hydroxylases. ACS Chem Biol 8(7):1488–1496

    Article  CAS  PubMed  Google Scholar 

  • Dehne N, Hintereder G, Brune B (2010) High glucose concentrations attenuate hypoxia-inducible factor-1alpha expression and signaling in non-tumor cells. Exp Cell Res 316(7):1179–1189

    Article  CAS  PubMed  Google Scholar 

  • Detmar M, Brown LF, Berse B et al (1997) Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin. J Invest Dermatol 108(3):263–268

    Article  CAS  PubMed  Google Scholar 

  • Egea V, von Baumgarten L, Schichor C et al (2011) TNF-alpha respecifies human mesenchymal stem cells to a neural fate and promotes migration toward experimental glioma. Cell Death Differ 18(5):853–863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fraisl P, Aragones J, Carmeliet P (2009) Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory disease. Nat Rev Drug Discov 8(2):139–152

    Article  CAS  PubMed  Google Scholar 

  • Graham JS, Chilcott RP, Rice P, Milner SM, Hurst CG, Maliner BI (2005) Wound healing of cutaneous sulfur mustard injuries: strategies for the development of improved therapies. J Burns Wounds 4:e1

    PubMed  PubMed Central  Google Scholar 

  • Graham JS, Stevenson RS, Mitcheltree LW et al (2009) Medical management of cutaneous sulfur mustard injuries. Toxicology 263(1):47–58

    Article  CAS  PubMed  Google Scholar 

  • Kallio PJ, Wilson WJ, O’Brien S, Makino Y, Poellinger L (1999) Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. J Biol Chem 274(10):6519–6525

    Article  CAS  PubMed  Google Scholar 

  • Kalucka J, Ettinger A, Franke K et al (2013) Loss of epithelial hypoxia-inducible factor prolyl hydroxylase 2 accelerates skin wound healing in mice. Mol Cell Biol 33(17):3426–3438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kehe K, Szinicz L (2005) Medical aspects of sulphur mustard poisoning. Toxicology 214(3):198–209

    Article  CAS  PubMed  Google Scholar 

  • Mace KA, Yu DH, Paydar KZ, Boudreau N, Young DM (2007) Sustained expression of Hif-1alpha in the diabetic environment promotes angiogenesis and cutaneous wound repair. Wound Repair Regen 15(5):636–645

    Article  PubMed  Google Scholar 

  • Martens ME, Smith WJ (2008) The role of NAD+ depletion in the mechanism of sulfur mustard-induced metabolic injury. Cutan Ocul Toxicol 27(1):41–53

    Article  CAS  PubMed  Google Scholar 

  • Martins VL, Caley M, O’Toole EA (2013) Matrix metalloproteinases and epidermal wound repair. Cell Tissue Res 351(2):255–268

    Article  CAS  PubMed  Google Scholar 

  • Moriyama M, Moriyama H, Uda J, Matsuyama A, Osawa M, Hayakawa T (2014) BNIP3 plays crucial roles in the differentiation and maintenance of epidermal keratinocytes. J Invest Dermatol 134(6):1627–1635

    Article  CAS  PubMed  Google Scholar 

  • Murray JK, Balan C, Allgeier AM et al (2010) Dipeptidyl-quinolone derivatives inhibit hypoxia inducible factor-1alpha prolyl hydroxylases-1, -2, and -3 with altered selectivity. J Comb Chem 12(5):676–686

    Article  CAS  PubMed  Google Scholar 

  • O’Toole EA, Marinkovich MP, Peavey CL et al (1997) Hypoxia increases human keratinocyte motility on connective tissue. J Clin Invest 100(11):2881–2891

    Article  PubMed  PubMed Central  Google Scholar 

  • Polzer H, Haasters F, Prall WC et al (2010) Quantification of fluorescence intensity of labeled human mesenchymal stem cells and cell counting of unlabeled cells in phase-contrast imaging: an open-source-based algorithm. Tissue Eng Part C Methods 16(6):1277–1285

    Article  PubMed  Google Scholar 

  • Popp T, Egea V, Kehe K et al (2011) Sulfur mustard induces differentiation in human primary keratinocytes: opposite roles of p38 and ERK1/2 MAPK. Toxicol Lett 204(1):43–51

    Article  CAS  PubMed  Google Scholar 

  • Rezvani HR, Ali N, Serrano-Sanchez M et al (2011) Loss of epidermal hypoxia-inducible factor-1alpha accelerates epidermal aging and affects re-epithelialization in human and mouse. J Cell Sci 124(Pt 24):4172–4183

    Article  CAS  PubMed  Google Scholar 

  • Ridgway PF, Ziprin P, Peck DH, Darzi AW (2005) Hypoxia increases reepithelialization via an alphavbeta6-dependent pathway. Wound Repair Regen 13(2):158–164

    Article  PubMed  Google Scholar 

  • Ries C, Egea V, Karow M, Kolb H, Jochum M, Neth P (2007) MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines. Blood 109(9):4055–4063

    Article  CAS  PubMed  Google Scholar 

  • Ries C, Popp T, Egea V, Kehe K, Jochum M (2009) Matrix metalloproteinase-9 expression and release from skin fibroblasts interacting with keratinocytes: upregulation in response to sulphur mustard. Toxicology 263(1):26–31

    Article  CAS  PubMed  Google Scholar 

  • Rossiter H, Barresi C, Pammer J et al (2004) Loss of vascular endothelial growth factor a activity in murine epidermal keratinocytes delays wound healing and inhibits tumor formation. Cancer Res 64(10):3508–3516

    Article  CAS  PubMed  Google Scholar 

  • Rowell M, Kehe K, Balszuweit F, Thiermann H (2009) The chronic effects of sulfur mustard exposure. Toxicology 263(1):9–11

    Article  CAS  PubMed  Google Scholar 

  • Ruthenborg RJ, Ban JJ, Wazir A, Takeda N, Kim JW (2014) Regulation of wound healing and fibrosis by hypoxia and hypoxia-inducible factor-1. Mol Cells 37(9):637–643

    Article  PubMed  PubMed Central  Google Scholar 

  • Saladi RN, Smith E, Persaud AN (2006) Mustard: a potential agent of chemical warfare and terrorism. Clin Exp Dermatol 31(1):1–5

    Article  CAS  PubMed  Google Scholar 

  • Salo T, Lyons JG, Rahemtulla F, Birkedal Hansen H, Larjava H (1991) Transforming growth factor-beta 1 up-regulates type IV collagenase expression in cultured human keratinocytes. J Biol Chem 266:11436–11441

    CAS  PubMed  Google Scholar 

  • Sanderson H, Fauser P, Thomsen M, Sorensen PB (2009) Human health risk screening due to consumption of fish contaminated with chemical warfare agents in the Baltic Sea. J Hazard Mater 162(1):416–422

    Article  CAS  PubMed  Google Scholar 

  • Scholz CC, Taylor CT (2013) Targeting the HIF pathway in inflammation and immunity. Curr Opin Pharmacol 13(4):646–653

    Article  CAS  PubMed  Google Scholar 

  • Semenza GL (2014) Oxygen sensing, hypoxia-inducible factors, and disease pathophysiology. Annu Rev Pathol 9:47–71

    Article  CAS  PubMed  Google Scholar 

  • Sen CK (2009) Wound healing essentials: let there be oxygen. Wound Repair Regen 17(1):1–18

    Article  PubMed  PubMed Central  Google Scholar 

  • Sugendran K, Jeevaratnam K, Husain K, Singh R, Srivastava DK (1992) Effects of topically applied sulphur mustard on tissue glycogen, blood glucose, lactate and pyruvate in mice. Indian J Physiol Pharmacol 36(3):219–221

    CAS  PubMed  Google Scholar 

  • Tian YM, Yeoh KK, Lee MK et al (2011) Differential sensitivity of hypoxia inducible factor hydroxylation sites to hypoxia and hydroxylase inhibitors. J Biol Chem 286(15):13041–13051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tracy K, Dibling BC, Spike BT, Knabb JR, Schumacker P, Macleod KF (2007) BNIP3 is an RB/E2F target gene required for hypoxia-induced autophagy. Mol Cell Biol 27(17):6229–6242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang X, Yan X, Cheng L et al (2013) Wound healing improvement with PHD-2 silenced fibroblasts in diabetic mice. PLoS One 8(12):e84548

    Article  PubMed  PubMed Central  Google Scholar 

  • Zimmermann AS, Morrison SD, Hu MS et al (2014) Epidermal or dermal specific knockout of PHD-2 enhances wound healing and minimizes ischemic injury. PLoS One 9(4):e93373

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was funded by grants from the Institute of Cardiovascular Prevention, Ludwig-Maximilians-University of Munich and by a contract of the German Armed Forces (M/SABX/BA003). We especially want to thank Thomas Pitsch for valuable technical assistance in C.R.’s laboratory, Christian Mahl for help with the analysis of the scratch wound assay, Donato Santovito for support in statistical analysis, and Mina Zarrabi as well as Ram Prasad for conducting the intoxication of cells at the Bundeswehr Institute of Pharmacology and Toxicology.

Conflict of interest

The authors declare no conflicts of interest.

Author information

Authors and Affiliations

  1. Institute for Cardiovascular Prevention, Ludwig-Maximilians-University of Munich, Pettenkoferstraße 9b, 80336, Munich, Germany

    Janina Deppe, Tanja Popp, Virginia Egea, Christian Weber & Christian Ries

  2. Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany

    Dirk Steinritz, Annette Schmidt & Horst Thiermann

  3. Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University, Munich, Germany

    Dirk Steinritz

  4. Molecular and Cellular Sport Medicine, German Sport University, Cologne, Germany

    Annette Schmidt

Authors
  1. Janina Deppe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tanja Popp
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Virginia Egea
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dirk Steinritz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Annette Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Horst Thiermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christian Weber
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christian Ries
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christian Ries.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Deppe, J., Popp, T., Egea, V. et al. Impairment of hypoxia-induced HIF-1α signaling in keratinocytes and fibroblasts by sulfur mustard is counteracted by a selective PHD-2 inhibitor. Arch Toxicol 90, 1141–1150 (2016). https://doi.org/10.1007/s00204-015-1549-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-015-1549-y

Keywords

Search

Navigation