Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective

Nature Medicine volume 22, pages 657–665 (2016)Cite this article

Subjects

Abstract

Although somatic cell activation of the embryonic stem cell (ESC) pluripotency factor OCT4 has been reported, this previous work has been controversial and has not demonstrated a functional role for OCT4 in somatic cells. Here we demonstrate that smooth muscle cell (SMC)-specific conditional knockout of Oct4 in Apoe−/− mice resulted in increased lesion size and changes in lesion composition that are consistent with decreased plaque stability, including a thinner fibrous cap, increased necrotic core area, and increased intraplaque hemorrhage. Results of SMC-lineage-tracing studies showed that these effects were probably the result of marked reductions in SMC numbers within lesions and SMC investment within the fibrous cap, which may result from impaired SMC migration. The reactivation of Oct4 within SMCs was associated with hydroxymethylation of the Oct4 promoter and was hypoxia inducible factor-1α (HIF-1α, encoded by HIF1A) and Krüppel-like factor-4 (KLF4)-dependent. These results provide the first direct evidence that OCT4 has a functional role in somatic cells, and they highlight the potential role of OCT4 in normal and diseased somatic cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Oct4 is activated in SMCs in atherosclerotic lesions and contributes to lesion pathogenesis.
Figure 2: SMC-specific conditional knockout of Oct4 increases multiple indices of atherosclerotic plaque instability.
Figure 3: SMC-specific, conditional knockout of Oct4 results in reduced numbers of lesional SMCs.
Figure 4: Loss of Oct4 within SMCs is associated with impaired SMC migration and reduced expression of Mmp3 and Mmp13, but not with any change in SMC apoptosis or proliferation.
Figure 5: Activation of the Oct4 promoter in vitro and in vivo is associated with increased hydroxymethylation.
Figure 6: Activation of Oct4 in SMCs is KLF4 and HIF-1α dependent.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Schöler, H.R., Ruppert, S., Suzuki, N., Chowdhury, K. & Gruss, P. New type of POU domain in germ line-specific protein Oct-4. Nature 344, 435–439 (1990).

    PubMed  Google Scholar 

  2. Lengner, C.J., Welstead, G.G. & Jaenisch, R. The pluripotency regulator Oct4: a role in somatic stem cells? Cell Cycle 7, 725–728 (2008).

    CAS  PubMed  Google Scholar 

  3. Firth, A.L., Yao, W., Remillard, C.V., Ogawa, A. & Yuan, J.X. Upregulation of Oct-4 isoforms in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension. Am. J. Physiol. Lung Cell. Mol. Physiol. 298, L548–L557 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. He, W., Li, K., Wang, F., Qin, Y.R. & Fan, Q.X. Expression of OCT4 in human esophageal squamous cell carcinoma is significantly associated with poorer prognosis. World J. Gastroenterol. 18, 712–719 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Holmberg, J. et al. Activation of neural and pluripotent stem cell signatures correlates with increased malignancy in human glioma. PLoS One 6, e18454 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Lengner, C.J. et al. Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell 1, 403–415 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Atlasi, Y., Mowla, S.J., Ziaee, S.A., Gokhale, P.J. & Andrews, P.W. OCT4 spliced variants are differentially expressed in human pluripotent and nonpluripotent cells. Stem Cells 26, 3068–3074 (2008).

    CAS  PubMed  Google Scholar 

  8. Takeda, J., Seino, S. & Bell, G.I. Human Oct3 gene family: cDNA sequences, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues. Nucleic Acids Res. 20, 4613–4620 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Cantz, T. et al. Absence of OCT4 expression in somatic tumor cell lines. Stem Cells 26, 692–697 (2008).

    CAS  PubMed  Google Scholar 

  10. Jez, M. et al. Expression and differentiation between OCT4A and its pseudogenes in human ESCs and differentiated adult somatic cells. PLoS One 9, e89546 (2014).

    PubMed  PubMed Central  Google Scholar 

  11. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    CAS  PubMed  Google Scholar 

  12. Buganim, Y. et al. Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell 150, 1209–1222 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Shu, J. et al. Induction of pluripotency in mouse somatic cells with lineage specifiers. Cell 153, 963–975 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Gao, Y. et al. Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming. Cell Stem Cell 12, 453–469 (2013).

    CAS  PubMed  Google Scholar 

  15. Owens, G.K., Kumar, M.S. & Wamhoff, B.R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol. Rev. 84, 767–801 (2004).

    CAS  PubMed  Google Scholar 

  16. Alexander, M.R. & Owens, G.K. Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease. Annu. Rev. Physiol. 74, 13–40 (2012).

    CAS  PubMed  Google Scholar 

  17. Libby, P. Inflammation in atherosclerosis. Nature 420, 868–874 (2002).

    CAS  PubMed  Google Scholar 

  18. Shankman, L.S.S. et al. KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis. Nat. Med. 21, 628–637 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Piedrahita, J.A., Zhang, S.H., Hagaman, J.R., Oliver, P.M. & Maeda, N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc. Natl. Acad. Sci. USA 89, 4471–4475 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Liedtke, S., Stephan, M. & Kögler, G. Oct4 expression revisited: potential pitfalls for data misinterpretation in stem cell research. Biol. Chem. 389, 845–850 (2008).

    CAS  PubMed  Google Scholar 

  21. Kehler, J. et al. Oct4 is required for primordial germ cell survival. EMBO Rep. 5, 1078–1083 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Wirth, A. et al. G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat. Med. 14, 64–68 (2008).

    CAS  PubMed  Google Scholar 

  23. Gomez, D., Shankman, L.S., Nguyen, A.T. & Owens, G.K. Detection of histone modifications at specific gene loci in single cells in histological sections. Nat. Methods 10, 171–177 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Nishimura, R.A. et al. 2014 AHA/ACC guideline for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J. Thorac. Cardiovasc. Surg. 148, e1–e132 (2014).

    PubMed  Google Scholar 

  25. Clausen, B.E., Burkhardt, C., Reith, W., Renkawitz, R. & Förster, I. Conditional gene targeting in macrophages and granulocytes using LysM-cre mice. Transgenic Res. 8, 265–277 (1999).

    CAS  PubMed  Google Scholar 

  26. Cherepanova, O.A. et al. Oxidized phospholipids induce type VIII collagen expression and vascular smooth muscle cell migration. Circ. Res. 104, 609–618 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Pidkovka, N.A. et al. Oxidized phospholipids induce phenotypic switching of vascular smooth muscle cells in vivo and in vitro. Circ. Res. 101, 792–801 (2007).

    CAS  PubMed  Google Scholar 

  28. Botquin, V. et al. New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2. Genes Dev. 12, 2073–2090 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Alexander, M.R. et al. Genetic inactivation of IL-1 signaling enhances atherosclerotic plaque instability and reduces outward vessel remodeling in advanced atherosclerosis in mice. J. Clin. Invest. 122, 70–79 (2012).

    CAS  PubMed  Google Scholar 

  30. Yang, S.W., Lim, L., Ju, S., Choi, D.H. & Song, H. Effects of matrix metalloproteinase 13 on vascular smooth muscle cells migration via Akt-ERK dependent pathway. Tissue Cell 47, 115–121 (2015).

    PubMed  Google Scholar 

  31. Mathieu, J. et al. HIF induces human embryonic stem cell markers in cancer cells. Cancer Res. 71, 4640–4652 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Marsch, E., Sluimer, J.C. & Daemen, M.J. Hypoxia in atherosclerosis and inflammation. Curr. Opin. Lipidol. 24, 393–400 (2013).

    CAS  PubMed  Google Scholar 

  33. Athanasiadou, R. et al. Targeting of de novo DNA methylation throughout the Oct-4 gene regulatory region in differentiating embryonic stem cells. PLoS One 5, e9937 (2010).

    PubMed  PubMed Central  Google Scholar 

  34. Feldman, N. et al. G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis. Nat. Cell Biol. 8, 188–194 (2006).

    CAS  PubMed  Google Scholar 

  35. Manabe, I. & Owens, G.K. Recruitment of serum response factor and hyperacetylation of histones at smooth muscle-specific regulatory regions during differentiation of a novel P19-derived in vitro smooth muscle differentiation system. Circ. Res. 88, 1127–1134 (2001).

    CAS  PubMed  Google Scholar 

  36. Cole, J.E. et al. Unexpected protective role for toll-like receptor 3 in the arterial wall. Proc. Natl. Acad. Sci. USA 108, 2372–2377 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Edfeldt, K., Swedenborg, J., Hansson, G.K. & Yan, Z.-Q. Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation 105, 1158–1161 (2002).

    CAS  PubMed  Google Scholar 

  38. Lee, J. et al. Activation of innate immunity is required for efficient nuclear reprogramming. Cell 151, 547–558 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Lambert, C.M., Roy, M., Robitaille, G.A., Richard, D.E. & Bonnet, S. HIF-1 inhibition decreases systemic vascular remodelling diseases by promoting apoptosis through a hexokinase 2-dependent mechanism. Cardiovasc. Res. 88, 196–204 (2010).

    CAS  PubMed  Google Scholar 

  40. Yoshida, T., Kaestner, K.H. & Owens, G.K. Conditional deletion of Krüppel-like factor 4 delays downregulation of smooth muscle cell differentiation markers but accelerates neointimal formation following vascular injury. Circ. Res. 102, 1548–1557 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Shatrov, V.A., Sumbayev, V.V., Zhou, J. & Brüne, B. Oxidized low-density lipoprotein (oxLDL) triggers hypoxia-inducible factor-1alpha (HIF-1α) accumulation via redox-dependent mechanisms. Blood 101, 4847–4849 (2003).

    CAS  PubMed  Google Scholar 

  42. Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106 (2008).

    CAS  PubMed  Google Scholar 

  43. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  PubMed  Google Scholar 

  44. Liao, Y., Smyth, G.K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    CAS  PubMed  Google Scholar 

  45. Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed  PubMed Central  Google Scholar 

  46. Huang, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    CAS  Google Scholar 

  47. Wiznerowicz, M. & Trono, D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J. Virol. 77, 8957–8961 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors would like to thank R. Tripathi, M. McCanna, T. Karaoli, T. Lillard and J. Sanders for their technical assistance; A. Newman, M. Quetsch, E. Schutzenhofer, J. Roithmayr and R. Haskins for their help with image analysis and cell culture experiments; S. Guillot at the Advanced Microscopy Facility at the University of Virginia for help with confocal microscopy; BioConnector Service, University of Virginia, for help with statistical analysis; D. Trono from Swiss Institutes of Technology, Lausanne, Switzerland, for the pLVTHM vector; H.R. Schöler from Max Plank Institute for the Oct4flox/flox mice; S. Offermanns from Max Plank Institute for the Myh11-CreERT2 mice; and G. Randolph for the LysMCre/Cre mice. This work was supported by US National Institutes of Health (NIH) grants R01 HL057353, R01 HL087867 and R01 HL098538 (to G.K.O.); AHA 11PRE17008 (to L.S.S.); Russian Science Foundation grant 14-14-00718 and Federal Agency of Scientific Organization (to A.T.); R01 HL100257 and R00 HL089412 (to J.J.C.); US Department of Defense Grant (W81XWH-10-2-0125, to Y.-J.G); AHA 13POST17080043 and 15SDG25860021 (to D.G.); AHA 11PRE7750030 (to M.M.); AHA 15PRE25670040 (to D.L.H.); and AHA 15PRE25730011 (to G.F.A.).

Author information

Authors and Affiliations

  1. Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia, USA

    Olga A Cherepanova, Delphine Gomez, Laura S Shankman, Pamela Swiatlowska, Gabriel F Alencar, Daniel L Hess, Melissa H Bevard, Elizabeth S Greene, Meera Murgai, Jessica J Connelly & Gary K Owens

  2. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA

    Delphine Gomez, Laura S Shankman & Gary K Owens

  3. Intercollegiate Faculty of Biotechnology, University of Gdansk, Gdansk, Poland

    Pamela Swiatlowska

  4. The Center for Cardiovascular Biology and Atherosclerosis Research, University of Texas Houston Medical School, Houston, Texas, USA

    Jason Williams & Yong-Jian Geng

  5. Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA

    Olga F Sarmento

  6. Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA

    Gabriel F Alencar, Daniel L Hess & Stefan Bekiranov

  7. Department of Pathology, University of Virginia, Charlottesville, Virginia, USA

    Meera Murgai

  8. Bioinformatics Core, School of Medicine, University of Virginia, Charlottesville, Virginia, USA

    Stephen D Turner

  9. Department of Medicine, University of Virginia, Charlottesville, Virginia, USA

    Jessica J Connelly

  10. Institute of Cytology, Russian Academy of Science, Saint Petersburg, Russia

    Alexey Tomilin

Authors
  1. Olga A Cherepanova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Delphine Gomez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laura S Shankman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pamela Swiatlowska
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jason Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Olga F Sarmento
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gabriel F Alencar
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Daniel L Hess
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Melissa H Bevard
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Elizabeth S Greene
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Meera Murgai
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Stephen D Turner
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yong-Jian Geng
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Stefan Bekiranov
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Jessica J Connelly
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Alexey Tomilin
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Gary K Owens
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

O.A.C. conducted most of the experiments, performed data analysis, generated most of the experimental mice and was the primary writer of the manuscript. D.G. performed ChIP assays, site-directed mutagenesis, bisulfite sequencing, in vitro hydroxymethylation analyses and ISH–PLA assays. L.S.S. generated SMC-lineage-tracing mice, performed immunostaining and image analysis. P.S. performed immunostaining and image analysis in the LysM-Cre mouse model. J.W. and Y.-J.G. performed in vivo hydroxymethylation analysis. O.F.S. performed in vitro experiments and bioinformatic analysis. G.F.A. and S.B. performed the RNA-seq analyses. D.L.H. performed the immunofluorescence and confocal microscopy for human samples. M.H.B. performed the immunohistochemistry. E.S.G. conducted statistical analyses and performed immunohistochemistry and data analysis. M.M. assisted in experiments with lentiviruses and was involved in necrotic core analysis. S.D.T. directed RNA-seq analysis and provided advice on statistical analysis. J.J.C. performed bioinformatic analysis and directed the methylation and hydroxymethylation analyses. A.T. generated the lentiviral plasmids and provided advice throughout the project. All authors participated in making final manuscript revisions. G.K.O. supervised the entire project and had a major role in experimental design, data interpretation, and writing the manuscript.

Corresponding author

Correspondence to Gary K Owens.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–3 (PDF 16269 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cherepanova, O., Gomez, D., Shankman, L. et al. Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective. Nat Med 22, 657–665 (2016). https://doi.org/10.1038/nm.4109

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4109

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing