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PKNOX2 expression and regulation in the bone marrow mesenchymal stem cells of Fanconi anemia patients and healthy donors

  • Ilgin Cagnan
  • Erdal Cosgun
  • Ozlen Konu
  • Duygu Uckan
  • Aysen Gunel-Ozcan
Original Article
  • 23 Downloads

Abstract

HOX and TALE transcription factors are important regulators of development and homeostasis in determining cellular identity. Deregulation of this process may drive cancer progression. The aim of this study was to investigate the expression of these transcription factors in the bone marrow derived mesenchymal stem cells (BM-MSCs) of Fanconi anemia (FA) patients, which is a cancer-predisposing disease. Expression levels of HOX and TALE genes in BM-MSCs were obtained from FA patients and healthy donors by RT-qPCR and highly conserved expression levels were observed between patient and donor cells, except PKNOX2, which is a member of TALE class. PKNOX2 was significantly downregulated in FA cells compared to donors (P < 0.05). PKNOX2 expression levels did not change with diepoxybutane (DEB), a DNA crosslinking agent, in either donor or FA cells except one patient’s with a truncation mutation of FANCA. A difference of PKNOX2 protein level was not obtained between FA patient and donor BM-MSCs by western blot analysis. When human TGF-β1 (rTGF-β1) recombinant protein was provided to the cultures, PKNOX2 as well as TGF-β1 expression increased both in FA and donor BM-MSCs in a dose dependent manner. 5 ng/mL rTGF-β stimulation had more dominant effect on the gene expression of donor BM-MSCs compared to FA cells. Decreased PKNOX2 expression in FA BM-MSCs may provide new insights into the molecular pathophysiology of the disease and TGF-β1 levels of the microenvironment may be the cause of PKNOX2 downregulation.

Keywords

PKNOX2 HOX genes TALE class TGF-β1 Fanconi anemia Bone marrow mesenchymal stem cells 

Notes

Acknowledgements

This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK; Project No: 110S021 in conjunction with EU COST Action BM0805 designated as ‘HOX and TALE transcription factors in Development and Disease’ and TUBITAK Project No: 214Z033). The data in this study is a part of Ilgin Cagnan’s Ph.D. thesis.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (PDF 7823 KB)
11033_2018_4522_MOESM2_ESM.pdf (114 kb)
Supplementary material 2 (PDF 114 KB)
11033_2018_4522_MOESM3_ESM.pdf (188 kb)
Supplementary material 3 (PDF 188 KB)

References

  1. 1.
    Illig R, Fritsch H, Schwarzer C (2013) Spatio-temporal expression of HOX genes in human hindgut development. Dev Dyn 242:53–66.  https://doi.org/10.1002/dvdy.23893 CrossRefPubMedGoogle Scholar
  2. 2.
    McGinnis W, Krumlauf R (1992) Homeobox genes and axial patterning. Cell 68:283–302CrossRefPubMedGoogle Scholar
  3. 3.
    Williamson I, Eskeland R, Lettice LA et al (2012) Anterior-posterior differences in HoxD chromatin topology in limb development. Development 139:3157–3167.  https://doi.org/10.1242/dev.081174 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Holland PW, Booth HA, Bruford EA (2007) Classification and nomenclature of all human homeobox genes. BMC Biol 5:47CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Merabet S, Mann RS (2016) To Be Specific or Not: The Critical Relationship Between Hox And TALE Proteins. Trends Genet 32:334–347.  https://doi.org/10.1016/j.tig.2016.03.004 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Iotti G, Longobardi E, Masella S et al (2011) Homeodomain transcription factor and tumor suppressor Prep1 is required to maintain genomic stability. Proc Natl Acad Sci U S A 108:E314–E322.  https://doi.org/10.1073/pnas.1105216108 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Thorsteinsdottir U, Kroon E, Jerome L, Blasi F, Sauvageau G (2001) Defining roles for HOX and MEIS1 genes in induction of acute myeloid leukemia. Mol Cell Biol 21:224–234CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ackema KB, Charite J (2008) Mesenchymal stem cells from different organs are characterized by distinct topographic Hox codes. Stem Cells Dev 17:979–991.  https://doi.org/10.1089/scd.2007.0220 CrossRefPubMedGoogle Scholar
  9. 9.
    Takahashi Y, Hamada J, Murakawa K et al (2004) Expression profiles of 39 HOX genes in normal human adult organs and anaplastic thyroid cancer cell lines by quantitative real-time RT-PCR system. Exp Cell Res 293:144–153CrossRefPubMedGoogle Scholar
  10. 10.
    Yamamoto M, Takai D, Yamamoto F (2003) Comprehensive expression profiling of highly homologous 39 hox genes in 26 different human adult tissues by the modified systematic multiplex RT-pCR method reveals tissue-specific expression pattern that suggests an important role of chromosomal structure in the regulation of Hox gene expression in adult tissues. Gene Expr 11:199–210CrossRefPubMedGoogle Scholar
  11. 11.
    Bhatlekar S, Fields JZ, Boman BM (2014) HOX genes and their role in the development of human cancers. J Mol Med 92:811–823.  https://doi.org/10.1007/s00109-014-1181-y CrossRefPubMedGoogle Scholar
  12. 12.
    Platais C, Hakami F, Darda L, Lambert DW, Morgan R, Hunter KD (2016) The role of HOX genes in head and neck squamous cell carcinoma. J Oral Pathol Med 45:239–247.  https://doi.org/10.1111/jop.12388 CrossRefPubMedGoogle Scholar
  13. 13.
    Bhattacharjee S, Nandi S (2018) Rare genetic diseases with defects in DNA repair: opportunities and challenges in orphan drug development for targeted cancer therapy. Cancers 10(298).  https://doi.org/10.3390/cancers10090298 CrossRefPubMedCentralGoogle Scholar
  14. 14.
    Hakem R (2008) DNA-damage repair; the good, the bad, and the ugly. EMBO J 27:589–605.  https://doi.org/10.1038/emboj.2008.15 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Mamrak NE, Shimamura A, Howlett NG (2016) Recent discoveries in the molecular pathogenesis of the inherited bone marrow failure syndrome Fanconi anemia. Blood Rev 31:93–99.  https://doi.org/10.1016/j.blre.2016.10.002 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Zhan-He W (2013) The concept and practice of Fanconi Anemia: from the clinical bedside to the laboratory bench. Transl Pediatr 2:112–119.  https://doi.org/10.3978/j.issn.2224-4336.2013.07.01 CrossRefGoogle Scholar
  17. 17.
    Knies K, Inano S, Ramirez MJ et al (2017) Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia. J Clin Invest 127:3013–3027.  https://doi.org/10.1172/JCI92069 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Auerbach AD (1993) Fanconi anemia diagnosis and the diepoxybutane (DEB) test. Exp Hematol 21:731–733PubMedGoogle Scholar
  19. 19.
    Zhang H, Kozono DE, O’Connor KW et al (2016) TGF-beta Inhibition rescues hematopoietic stem cell defects and bone marrow failure in Fanconi anemia. Cell Stem Cell 18:668–681.  https://doi.org/10.1016/j.stem.2016.03.002 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wang N, Kim HG, Cotta CV et al (2006) TGFbeta/BMP inhibits the bone marrow transformation capability of Hoxa9 by repressing its DNA-binding ability. EMBO J 25:1469–1480CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Walsh CM, Carroll SB (2007) Collaboration between Smads and a Hox protein in target gene repression. Development 134:3585–3592CrossRefPubMedGoogle Scholar
  22. 22.
    Ruiz i Altaba A, Melton DA (1989) Interaction between peptide growth factors and homoeobox genes in the establishment of antero-posterior polarity in frog embryos. Nature 341:33–38CrossRefPubMedGoogle Scholar
  23. 23.
    Kloen P, Visker MH, Olijve W, van Zoelen EJ, Boersma CJ (1997) Cell-type-specific modulation of Hox gene expression by members of the TGF-beta superfamily: a comparison between human osteosarcoma and neuroblastoma cell lines. Biochem Biophys Res Commun 233:365–369CrossRefPubMedGoogle Scholar
  24. 24.
    Cagnan I (2018) HOX and TALE transcription factors in Fanconi anemia bone-marrow mesenchymal stem cells: gene expression and protein interactions. Dissertation, Hacettepe UniversityGoogle Scholar
  25. 25.
    Cagnan I, Gunel-Ozcan A, Aerts-Kaya F et al (2018) Bone marrow mesenchymal stem cells carrying FANCD2 mutation differ from the other Fanconi anemia complementation groups in terms of TGF-beta1 production. Stem Cell Rev 14:425–437.  https://doi.org/10.1007/s12015-017-9794-5 CrossRefPubMedGoogle Scholar
  26. 26.
    Cagnan I, Aerts Kaya F, Cetinkaya D, Gunel Ozcan G (2017) Stably expressed reference genes during differentiation of bone marrow-derived mesenchymal stromal cells. Turkish J Biol 41:88–97.  https://doi.org/10.3906/biy-1511-93 CrossRefGoogle Scholar
  27. 27.
    Van Buuren S, Groothuis-Oudshoorn K (2011) mice: Multivariate Imputation by Chained Equations in R. J Statistical Software 45:1–67.  https://doi.org/10.18637/jss.v045.i03 CrossRefGoogle Scholar
  28. 28.
    Azur MJ, Stuart EA, Frangakis C, Leaf PJ (2011) Multiple imputation by chained equations: what is it and how does it work? Int J Methods Psychiatr Res 20:40–49.  https://doi.org/10.1002/mpr.329 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Waljee AK, Mukherjee A, Singal AG et al (2013) Comparison of imputation methods for missing laboratory data in medicine. BMJ Open 3.  https://doi.org/10.1136/bmjopen-2013-002847 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Davarinejad H (2017) Quantifications of western blots with Image J. http://www.yorku.ca/yisheng/Internal/Protocols/ImageJ.pdf. (Accessed 01 October 2017)
  31. 31.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(− delta delta C(T)) method. Methods 25:402–408CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Liedtke S, Buchheiser A, Bosch J et al (2010) The HOX Code as a “biological fingerprint” to distinguish functionally distinct stem cell populations derived from cord blood. Stem Cell Res 5:40–50.  https://doi.org/10.1016/j.scr.2010.03.004 CrossRefPubMedGoogle Scholar
  34. 34.
    Picchi J, Trombi L, Spugnesi L et al (2013) HOX and TALE signatures specify human stromal stem cell populations from different sources. J Cell Physiol 228:879–889.  https://doi.org/10.1002/jcp.24239 CrossRefPubMedGoogle Scholar
  35. 35.
    Kim M, Hwang S, Park K, Kim SY, Lee YK, Lee DS (2015) Increased expression of interferon signaling genes in the bone marrow microenvironment of myelodysplastic syndromes. PLoS ONE 10:e0120602.  https://doi.org/10.1371/journal.pone.0120602 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Ramos TL, Sánchez-Abarca LI, Rosón-Burgo B et al (2017) Mesenchymal stromal cells (MSC) from JAK2 + myeloproliferative neoplasms differ from normal MSC and contribute to the maintenance of neoplastic hematopoiesis. PLoS ONE 12:e0182470.  https://doi.org/10.1371/journal.pone.0182470 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Pittenger MF, Mackay AM, Beck SC et al (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Baxter M, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I (2004) Study of telomere length reveal rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 22:675–682CrossRefPubMedGoogle Scholar
  39. 39.
    Wagner W, Horn P, Castoldi M, Diehlmann A, Bork S, Saffrich R, Benes V, Blake J, Pfiser S, Ecstein V et al (2008) Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS ONE 3.  https://doi.org/10.1371/journal.pone.0002213 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Xu J, Li X, Cole A, Sherman Z, Du W (2018) Reduced cell division control protein 42 activity compromises hematopoiesis-supportive function of Fanconi anemia mesenchymal stromal cells. Stem Cells 36:785–795.  https://doi.org/10.1002/stem.2789 CrossRefPubMedGoogle Scholar
  41. 41.
    Greenbaum D, Colangelo C, Williams K, Gerstein M (2003) Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol 4:117CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Fognani C, Kilstrup-Nielsen C, Berthelsen J, Ferretti E, Zappavigna V, Blasi F (2002) Characterization of PREP2, a paralog of PREP1, which defines a novel sub-family of the MEINOX TALE homeodomain transcription factors. Nucleic Acids Res 30:2043–2051CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Haller K, Rambaldi I, Daniels E, Featherstone M (2004) Subcellular localization of multiple PREP2 isoforms is regulated by actin, tubulin, and nuclear export. J Biol Chem 279:49384–49394CrossRefPubMedGoogle Scholar
  44. 44.
    Zhou W, Zhu H, Zhao J et al (2013) Misexpression of Pknox2 in mouse limb bud mesenchyme perturbs zeugopod development and deltoid crest formation. PloS ONE 8:e64237.  https://doi.org/10.1371/journal.pone.0064237 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Hurov KE, Cotta-Ramusino C, Elledge SJ (2010) A genetic screen identifies the Triple T complex required for DNA damage signaling and ATM and ATR stability. Genes Dev 24:1939–1950.  https://doi.org/10.1101/gad.1934210 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Lambert SA, Jolma A, Campitelli LF et al (2018) The human transcription factors. Cell 172:650–665.  https://doi.org/10.1016/j.cell.2018.01.029 CrossRefPubMedGoogle Scholar
  47. 47.
    Wu J, Niu J, Li X, Wang X, Guo Z, Zhang F (2014) TGF-β1 induces senescence of bone marrow mesenchymal stem cells via increase of mitochondrial ROS production. BMC Dev Biol 14:21.  https://doi.org/10.1186/1471-213X-14-21 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Schedel J, Lowin T, Kujat R et al (2010) RAP-PCR fingerprinting reveals time-dependent expression of matrix-related molecules following stem-cell based TGFβ1-induced chondrocyte development. Int J Mol Med 27:519–523.  https://doi.org/10.3892/ijmm.2011.608 CrossRefGoogle Scholar
  49. 49.
    Walenda G, Abnaof K, Joussen S et al (2013) TGF-beta1 does not induce senescence of multipotent mesenchymal stromal cells and has similar effects in early and late passages. PloS ONE 8:e77656.  https://doi.org/10.1371/journal.pone.0077656 CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Dardaei L, Longobardi E, Blasi F (2014) Prep1 and Meis1 competition for Pbx1 binding regulates protein stability and tumorigenesis. Proc Natl Acad Sci U S A 111:E896–E905.  https://doi.org/10.1073/pnas.1321200111 CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Li J, Tripathi BJ, Chalam KV, Tripathi RC (1996) Transforming growth factor-beta 1 and -beta 2 positively regulate TGF-beta 1 mRNA expression in trabecular cells. Invest Ophthalmol Vis Sci 37:2778–2782PubMedGoogle Scholar
  52. 52.
    Bakker ST, de Winter JP, te Riele H (2013) Learning from a paradox: recent insight into Fanconi anaemia through studying mouse models. Dis Model Mech 6:40–47.  https://doi.org/10.1002/stem.2789 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Stem Cell Sciences, Graduate School of Health Sciences, Center for Stem Cell Research and DevelopmentHacettepe UniversityAnkaraTurkey
  2. 2.Department of Biostatistics, Faculty of MedicineHacettepe UniversityAnkaraTurkey
  3. 3.Department of Molecular Biology and GeneticsBilkent UniversityAnkaraTurkey
  4. 4.Department of Pediatrics, Division of Bone Marrow Transplantation Unit, Faculty of MedicineHacettepe UniversityAnkaraTurkey
  5. 5.Blood Bank, Burhan Nalbantoglu State HospitalNicosiaNorth Cyprus
  6. 6.Microsoft ResearchRedmondUSA

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