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:

Salt-sensitive hypertension in circadian clock–deficient Cry-null mice involves dysregulated adrenal Hsd3b6

Nature Medicine volume 16, pages 67–74 (2010)Cite this article

Abstract

Malfunction of the circadian clock has been linked to the pathogenesis of a variety of diseases. We show that mice lacking the core clock components Cryptochrome-1 (Cry1) and Cryptochrome-2 (Cry2) (Cry-null mice) show salt-sensitive hypertension due to abnormally high synthesis of the mineralocorticoid aldosterone by the adrenal gland. An extensive search for the underlying cause led us to identify type VI 3β-hydroxyl-steroid dehydrogenase (Hsd3b6) as a new hypertension risk factor in mice. Hsd3b6 is expressed exclusively in aldosterone-producing cells and is under transcriptional control of the circadian clock. In Cry-null mice, Hsd3b6 messenger RNA and protein levels are constitutively high, leading to a marked increase in 3β-hydroxysteroid dehydrogenase-isomerase (3β-HSD) enzymatic activity and, as a consequence, enhanced aldosterone production. These data place Hsd3b6 in a pivotal position through which circadian clock malfunction is coupled to the development of hypertension. Translation of these findings to humans will require clinical examination of human HSD3B1 gene, which we found to be functionally similar to mouse Hsd3b6.

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: Overproduction of aldosterone by Cry-null adrenal glands.
Figure 2: Chronically high expression of Hsd3b6 in aldosterone-producing zona glomerulosa (ZG) cells of Cry-null adrenal glands.
Figure 3: Coexpression of Hsd3b6 protein and Cyp11b2 mRNA in adrenal zona glomerulosa cells.
Figure 4: Elevated 3β-HSD activity is responsible for aldosterone overproduction in Cry-null mice.
Figure 5: Cry-null mice show salt-sensitive hypertension.
Figure 6: Identification of a zona glomerulosa–specific HSD3B isoform in the human adrenal gland.

Similar content being viewed by others

References

  1. Staessen, J.A., Wang, J., Bianchi, G. & Birkenhager, W.H. Essential hypertension. Lancet 361, 1629–1641 (2003).

    Article  Google Scholar 

  2. Lifton, R.P., Gharavi, A.G. & Geller, D.S. Molecular mechanisms of human hypertension. Cell 104, 545–556 (2001).

    Article  CAS  Google Scholar 

  3. Schibler, U. & Sassone-Corsi, P. A web of circadian pacemakers. Cell 111, 919–922 (2002).

    Article  CAS  Google Scholar 

  4. Dunlap, J.C. Molecular bases for circadian clocks. Cell 96, 271–290 (1999).

    Article  CAS  Google Scholar 

  5. Reppert, S.M. & Weaver, D.R. Coordination of circadian timing in mammals. Nature 418, 935–941 (2002).

    Article  CAS  Google Scholar 

  6. Wijnen, H. & Young, M.W. Interplay of circadian clocks and metabolic rhythms. Annu. Rev. Genet. 40, 409–448 (2006).

    Article  CAS  Google Scholar 

  7. Takahashi, J.S., Hong, H.K., Ko, C.H. & McDearmon, E.L. The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat. Rev. Genet. 9, 764–775 (2008).

    Article  CAS  Google Scholar 

  8. Panda, S. et al. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 109, 307–320 (2002).

    Article  CAS  Google Scholar 

  9. Green, C.B., Takahashi, J.S. & Bass, J. The meter of metabolism. Cell 134, 728–742 (2008).

    Article  CAS  Google Scholar 

  10. Hastings, M.H., Reddy, A.B. & Maywood, E.S. A clockwork web: circadian timing in brain and periphery, in health and disease. Nat. Rev. Neurosci. 4, 649–661 (2003).

    Article  CAS  Google Scholar 

  11. Furlan, R. et al. Modifications of cardiac autonomic profile associated with a shift schedule of work. Circulation 102, 1912–1916 (2000).

    Article  CAS  Google Scholar 

  12. Bradley, T.D. & Floras, J.S. Sleep apnea and heart failure: Part II: central sleep apnea. Circulation 107, 1822–1826 (2003).

    Article  Google Scholar 

  13. Scheer, F.A., Hilton, M.F., Mantzoros, C.S. & Shea, S.A. Adverse metabolic and cardiovascular consequences of circadian misalignment. Proc. Natl. Acad. Sci. USA 106, 4453–4458 (2009).

    Article  CAS  Google Scholar 

  14. van der Horst, G.T. et al. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature 398, 627–630 (1999).

    Article  CAS  Google Scholar 

  15. Vitaterna, M.H. et al. Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2. Proc. Natl. Acad. Sci. USA 96, 12114–12119 (1999).

    Article  CAS  Google Scholar 

  16. Matsuo, T. et al. Control mechanism of the circadian clock for timing of cell division in vivo. Science 302, 255–259 (2003).

    Article  CAS  Google Scholar 

  17. Yamaguchi, S. et al. Role of DBP in the circadian oscillatory mechanism. Mol. Cell. Biol. 20, 4773–4781 (2000).

    Article  CAS  Google Scholar 

  18. Kume, K. et al. mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98, 193–205 (1999).

    Article  CAS  Google Scholar 

  19. Okamura, H. et al. Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock. Science 286, 2531–2534 (1999).

    Article  CAS  Google Scholar 

  20. Mitsui, S., Yamaguchi, S., Matsuo, T., Ishida, Y. & Okamura, H. Antagonistic role of E4BP4 and PAR proteins in the circadian oscillatory mechanism. Genes Dev. 15, 995–1006 (2001).

    Article  CAS  Google Scholar 

  21. Ishida, A. et al. Light activates the adrenal gland: timing of gene expression and glucocorticoid release. Cell Metab. 2, 297–307 (2005).

    Article  CAS  Google Scholar 

  22. Balsalobre, A. et al. Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science 289, 2344–2347 (2000).

    Article  CAS  Google Scholar 

  23. Young, W.F. Primary aldosteronism: renaissance of a syndrome. Clin. Endocrinol. 66, 607–618 (2007).

    Article  CAS  Google Scholar 

  24. Kaplan, N.M. Primary aldosteronism. in Clinical Hypertension 410–433 (Lippincott Williams & Wilkins, Philadelphia, 2006).

  25. Brown, N.J. Eplerenone: cardiovascular protection. Circulation 107, 2512–2518 (2003).

    Article  CAS  Google Scholar 

  26. Gomez-Sanchez, C., Holland, O.B., Higgins, J.R., Kem, D.C. & Kaplan, N.M. Circadian rhythms of serum renin activity and serum corticosterone, prolactin and aldosterone concentrations in the male rat on normal and low-sodium diets. Endocrinology 99, 567–572 (1976).

    Article  CAS  Google Scholar 

  27. Simard, J. et al. Molecular biology of the 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase gene family. Endocr. Rev. 26, 525–582 (2005).

    Article  CAS  Google Scholar 

  28. Payne, A.H. & Hales, D.B. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr. Rev. 25, 947–970 (2004).

    Article  CAS  Google Scholar 

  29. Giroud, C.J., Stachenko, J. & Venning, E.H. Secretion of aldosterone by the zona glomerulosa of rat adrenal glands incubated in vitro. Proc. Soc. Exp. Biol. Med. 92, 154–158 (1956).

    Article  CAS  Google Scholar 

  30. Domalik, L.J. et al. Different isozymes of mouse 11 β-hydroxylase produce mineralocorticoids and glucocorticoids. Mol. Endocrinol. 5, 1853–1861 (1991).

    Article  CAS  Google Scholar 

  31. Ogishima, T., Suzuki, H., Hata, J., Mitani, F. & Ishimura, Y. Zone-specific expression of aldosterone synthase cytochrome P-450 and cytochrome P-45011β in rat adrenal cortex: histochemical basis for the functional zonation. Endocrinology 130, 2971–2977 (1992).

    Article  CAS  Google Scholar 

  32. Rainey, W.E. Adrenal zonation: clues from 11β-hydroxylase and aldosterone synthase. Mol. Cell. Endocrinol. 151, 151–160 (1999).

    Article  CAS  Google Scholar 

  33. Potts, G.O., Creange, J.E., Hardomg, H.R. & Schane, H.P. Trilostane, an orally active inhibitor of steroid biosynthesis. Steroids 32, 257–267 (1978).

    Article  CAS  Google Scholar 

  34. Jungmann, E. et al. The inhibiting effect of trilostane on adrenal steroid synthesis: hormonal and morphological alterations induced by subchronic trilostane treatment in normal rats. Res. Exp. Med. (Berl.) 180, 193–200 (1982).

    Article  CAS  Google Scholar 

  35. Brown, R., Quirk, J. & Kirkpatrick, P. Eplerenone. Nat. Rev. Drug Discov. 2, 177–178 (2003).

    Article  CAS  Google Scholar 

  36. Mason, J.I. et al. The regulation of 3β-hydroxysteroid dehydrogenase expression. Steroids 62, 164–168 (1997).

    Article  CAS  Google Scholar 

  37. Makhanova, N., Hagaman, J., Kim, H.S. & Smithies, O. Salt-sensitive blood pressure in mice with increased expression of aldosterone synthase. Hypertension 51, 134–140 (2008).

    Article  CAS  Google Scholar 

  38. Rhéaume, E. et al. Structure and expression of a new complementary DNA encoding the almost exclusive 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase in human adrenal glands and gonads. Mol. Endocrinol. 5, 1147–1157 (1991).

    Article  Google Scholar 

  39. Abbaszade, I.G. et al. Isolation of a new mouse 3β-hydroxysteroid dehydrogenase isoform, 3β-HSD VI, expressed during early pregnancy. Endocrinology 138, 1392–1399 (1997).

    Article  CAS  Google Scholar 

  40. Moisan, A.M. et al. New insight into the molecular basis of 3β-hydroxysteroid dehydrogenase deficiency: identification of eight mutations in the HSD3B2 gene eleven patients from seven new families and comparison of the functional properties of twenty-five mutant enzymes. J. Clin. Endocrinol. Metab. 84, 4410–4425 (1999).

    CAS  PubMed  Google Scholar 

  41. Rhéaume, E. et al. Congenital adrenal hyperplasia due to point mutations in the type II 3β-hydroxysteroid dehydrogenase gene. Nat. Genet. 1, 239–245 (1992).

    Article  Google Scholar 

  42. Peng, L., Arensburg, J., Orly, J. & Payne, A.H. The murine 3β-hydroxysteroid dehydrogenase (3β-HSD) gene family: a postulated role for 3β-HSD VI during early pregnancy. Mol. Cell. Endocrinol. 187, 213–221 (2002).

    Article  CAS  Google Scholar 

  43. Nakada, T. et al. Primary aldosteronism treated by trilostane (3β-hydroxysteroid dehydrogenase inhibitor). Urology 25, 207–214 (1985).

    Article  CAS  Google Scholar 

  44. Winterberg, B., Vetter, W., Groth, H., Greminger, P. & Vetter, H. Primary aldosteronism: treatment with trilostane. Cardiology 72 (Suppl 1), 117–121 (1985).

    Article  Google Scholar 

  45. Maeda, A. et al. Circadian intraocular pressure rhythm is generated by clock genes. Invest. Ophthalmol. Vis. Sci. 47, 4050–4052 (2006).

    Article  Google Scholar 

  46. Matsunaga, M., Ukena, K., Baulieu, E.E. & Tsutsui, K. 7α-hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc. Natl. Acad. Sci. USA 101, 17282–17287 (2004).

    Article  CAS  Google Scholar 

  47. Kutyavin, I.V. et al. 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 28, 655–661 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Ono, A. Hirasawa, T. Koshimizu, K. Terasawa, H. Nishinaga, M. Sato, Y. Yamaguchi, M. Matsuo, J.M. Fustin, K. Toida, H. Sei and K. Ishimura for technical support and valuable discussion. We also thank T. Michel for critical reading of the manuscript. This work was supported in part by the Specially Promoted Research (to H.O.) and Grant-in-Aid for Young Scientists (to M.D.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and grants from Nakatomi Foundation, SRF (to H.O.), Senri Life Science Foundation, Takeda Science Foundation (to M.D.) and the Netherlands Organization of Scientific research, ZonMW Vici 918.36.619 (to G.T.J.v.d.H.). Trilostane was a generous gift from Mochida Pharmaceutical. Eplerenone was a generous gift from Pfizer.

Author information

Authors and Affiliations

  1. Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.,

    Masao Doi, Yukari Takahashi, Rie Komatsu, Fumiyoshi Yamazaki, Hiroyuki Yamada & Hitoshi Okamura

  2. Department of Biology, Laboratory of Integrative Brain Sciences, Faculty of Education and Integrated Arts and Sciences, Waseda University, Shinjuku-ku, Tokyo, Japan.,

    Shogo Haraguchi & Kazuyoshi Tsutsui

  3. Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Chuo-ku, Kobe, Japan.,

    Noriaki Emoto

  4. Department of Systems Bioscience for Drug Discovery, Kyoto University, Sakyo-ku, Kyoto, Japan.,

    Yasushi Okuno

  5. Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.,

    Gozoh Tsujimoto

  6. Department of Urology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan.,

    Akihiro Kanematsu & Osamu Ogawa

  7. Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, Yamadaoka, Suita, Japan.,

    Takeshi Todo

  8. Department of Genetics, Center for Biomedical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands

    Gijsbertus T J van der Horst

Authors
  1. Masao Doi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yukari Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rie Komatsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fumiyoshi Yamazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hiroyuki Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shogo Haraguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Noriaki Emoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yasushi Okuno
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gozoh Tsujimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Akihiro Kanematsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Osamu Ogawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Takeshi Todo
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Kazuyoshi Tsutsui
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Gijsbertus T J van der Horst
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Hitoshi Okamura
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.D. and H.O. designed the research; A.K., O.O., T.T. and G.T.J.v.d.H. supplied the experimental materials; M.D., Y.T., R.K., F.Y., H.Y., S.H., K.T. and H.O. acquired the data; M.D., N.E., Y.O., G.T., K.T. and H.O. analyzed the data; and M.D., G.T.J.v.d.H. and H.O. drafted the manuscript.

Corresponding author

Correspondence to Hitoshi Okamura.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–3 and Supplementary Methods (PDF 1570 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Doi, M., Takahashi, Y., Komatsu, R. et al. Salt-sensitive hypertension in circadian clock–deficient Cry-null mice involves dysregulated adrenal Hsd3b6. Nat Med 16, 67–74 (2010). https://doi.org/10.1038/nm.2061

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.2061

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