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Enhancement of Flow-Induced AP-1 Gene Expression by Cyclosporin A Requires NFAT-Independent Signaling in Bone Cells

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Abstract

Growing evidence suggests that aging compromises the ability of the skeleton to respond to anabolic mechanical stimuli. Recently, we reported that treating senescent mice with Cyclosporin A (CsA) rescued aging-related deficits in loading-induced bone formation. Given that the actions of CsA are often attributed to inhibition of the calcineurin/NFAT axis, we hypothesized that CsA enhances gene expression in bone cells exposed to fluid flow, by inhibiting nuclear NFATc1 accumulation. When exposed to flow, MC3T3-E1 osteoblastic cells exhibited rapid nuclear accumulation of NFATc1 that was abolished by CsA treatment. Under differentiation conditions, intermittent CsA treatment enhanced gene expression of late osteoblastic differentiation markers and activator protein 1 (AP-1) family members. Superimposing flow upon CsA further enhanced expression of the AP-1 members Fra-1 and c-Jun. To delineate the contribution of NFAT in this response, cells were treated with VIVIT, a specific inhibitor of the calcineurin/NFAT interaction. Treatment with VIVIT blocked flow-induced nuclear NFATc1 accumulation but did not recapitulate the CsA-mediated enhancement of flow-induced AP-1 component gene expression. Taken together, our study is the first to demonstrate that CsA enhances mechanically-induced gene expression of AP-1 components in bone cells, and suggests that this response requires calcineurin-dependent mechanisms that are independent of inhibiting NFATc1 nuclear accumulation.

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Abbreviations

AP-1:

Activator protein-1

NFAT:

Nuclear factor of activated T cells

CsA:

Cyclosporin A

GSK-3β:

Glycogen synthase kinase-3β

Elk-1:

ETS-like transcription factor 1

NF-κB:

Nuclear factor κB

CREB:

Cyclic AMP response element-binding protein

ALP:

Alkaline phosphatase

OPN:

Osteopontin

OCN:

Osteocalcin

Cox2:

Cyclooxygenase 2

References

  1. Akhouayri, O., and R. St-Arnaud. Differential mechanisms of transcriptional regulation of the mouse osteocalcin gene by Jun family members. Calcif. Tissue Int. 80:123–131, 2007.

    Article  Google Scholar 

  2. Alles, N., N. S. Soysa, J. Hayashi, M. Khan, A. Shimoda, H. Shimokawa, O. Ritzeler, K. Akiyoshi, K. Aoki, and K. Ohya. Suppression of NF-kappaB increases bone formation and ameliorates osteopenia in ovariectomized mice. Endocrinology 151:4626–4634, 2010.

    Article  Google Scholar 

  3. Aramburu, J., A. Rao, and C. B. Klee. Calcineurin: from structure to function. Curr. Top. Cell Regul. 36:237–295, 2000.

    Article  Google Scholar 

  4. Aramburu, J., M. B. Yaffe, C. Lopez-Rodriguez, L. C. Cantley, P. G. Hogan, and A. Rao. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science 285:2129–2133, 1999.

    Article  Google Scholar 

  5. Asanuma, M., N. Ogawa, S. Nishibayashi, Y. Kondo, and A. Mori. Effects of repeated injection of cyclosporin A on pentylenetetrazol-induced convulsion and cyclophilin mRNA levels in rat brain. Neurochem. Res. 20:101–105, 1995.

    Article  Google Scholar 

  6. Behrens, A., J. Haigh, F. Mechta-Grigoriou, A. Nagy, M. Yaniv, and E. F. Wagner. Impaired intervertebral disc formation in the absence of Jun. Development 130:103–109, 2003.

    Article  Google Scholar 

  7. Bito, H., K. Deisseroth, and R. W. Tsien. CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87:1203–1214, 1996.

    Article  Google Scholar 

  8. Celil Aydemir, A. B., S. Lee, D. Won Kim, T. R. Gardner, D. Prince, J. Mok Ahn, and F. Y. Lee. Nuclear factor of activated T cell mediates proinflammatory gene expression in response to mechanotransduction. Ann. N Y Acad. Sci. 1117:138–142, 2007.

    Article  Google Scholar 

  9. Celil Aydemir, A. B., H. Minematsu, T. R. Gardner, K. O. Kim, J. M. Ahn, and F. Y. Lee. Nuclear factor of activated T cells mediates fluid shear stress- and tensile strain-induced Cox2 in human and murine bone cells. Bone 46:167–175, 2010.

    Article  Google Scholar 

  10. Chang, J., Z. Wang, E. Tang, Z. Fan, L. McCauley, R. Franceschi, K. Guan, P. H. Krebsbach, and C. Y. Wang. Inhibition of osteoblastic bone formation by nuclear factor-kappaB. Nat. Med. 15:682–689, 2009.

    Article  Google Scholar 

  11. Cheung, A. M., and L. Giangregorio. Mechanical stimuli and bone health: what is the evidence? Curr. Opin. Rheumatol. 24:561–566, 2012.

    Article  Google Scholar 

  12. D’Alonzo, R. C., N. Selvamurugan, G. Karsenty, and N. C. Partridge. Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation. J. Biol. Chem. 277:816–822, 2002.

    Article  Google Scholar 

  13. Donahue, T. L., T. R. Haut, C. E. Yellowley, H. J. Donahue, and C. R. Jacobs. Mechanosensitivity of bone cells to oscillating fluid flow induced shear stress may be modulated by chemotransport. J. Biomech. 36:1363–1371, 2003.

    Article  Google Scholar 

  14. Donahue, S. W., C. R. Jacobs, and H. J. Donahue. Flow-induced calcium oscillations in rat osteoblasts are age, loading frequency, and shear stress dependent. Am. J. Physiol. Cell Physiol. 281:C1635–C1641, 2001.

    Google Scholar 

  15. Eferl, R., A. Hoebertz, A. F. Schilling, M. Rath, F. Karreth, L. Kenner, M. Amling, and E. F. Wagner. The Fos-related antigen Fra-1 is an activator of bone matrix formation. EMBO J. 23:2789–2799, 2004.

    Article  Google Scholar 

  16. Flanagan, W. M., B. Corthesy, R. J. Bram, and G. R. Crabtree. Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352:803–807, 1991.

    Article  Google Scholar 

  17. Frischbutter, S., C. Gabriel, H. Bendfeldt, A. Radbruch, and R. Baumgrass. Dephosphorylation of Bcl-10 by calcineurin is essential for canonical NF-kappaB activation in Th cells. Eur. J. Immunol. 41:2349–2357, 2011.

    Article  Google Scholar 

  18. Grigoriadis, A. E., K. Schellander, Z. Q. Wang, and E. F. Wagner. Osteoblasts are target cells for transformation in c-fos transgenic mice. J. Cell Biol. 122:685–701, 1993.

    Article  Google Scholar 

  19. Hipskind, R. A., and G. Bilbe. MAP kinase signaling cascades and gene expression in osteoblasts. Front Biosci 3:d804–d816, 1998.

    Google Scholar 

  20. Inoue, D., S. Kido, and T. Matsumoto. Transcriptional induction of FosB/DeltaFosB gene by mechanical stress in osteoblasts. J. Biol. Chem. 279:49795–49803, 2004.

    Article  Google Scholar 

  21. Jochum, W., J. P. David, C. Elliott, A. Wutz, H. Plenk, Jr., K. Matsuo, and E. F. Wagner. Increased bone formation and osteosclerosis in mice overexpressing the transcription factor Fra-1. Nat. Med. 6:980–984, 2000.

    Article  Google Scholar 

  22. Julien, M., S. Khoshniat, A. Lacreusette, M. Gatius, A. Bozec, E. F. Wagner, Y. Wittrant, M. Masson, P. Weiss, L. Beck, D. Magne, and J. Guicheux. Phosphate-dependent regulation of MGP in osteoblasts: role of ERK1/2 and Fra-1. J. Bone Miner. Res. 24:1856–1868, 2009.

    Article  Google Scholar 

  23. Kim, Y., Y. I. Lee, M. Seo, S. Y. Kim, J. E. Lee, H. D. Youn, Y. S. Kim, and Y. S. Juhnn. Calcineurin dephosphorylates glycogen synthase kinase-3 beta at serine-9 in neuroblast-derived cells. J. Neurochem. 111:344–354, 2009.

    Article  Google Scholar 

  24. Kwon, R. Y., and C. R. Jacobs. Time-dependent deformations in bone cells exposed to fluid flow in vitro: investigating the role of cellular deformation in fluid flow-induced signaling. J. Biomech. 40:3162–3168, 2007.

    Article  Google Scholar 

  25. Kwon, R. Y., D. R. Meays, A. S. Meilan, J. Jones, R. Miramontes, N. Kardos, J. C. Yeh, and J. A. Frangos. Skeletal adaptation to intramedullary pressure-induced interstitial fluid flow is enhanced in mice subjected to targeted osteocyte ablation. PLoS ONE 7:e33336, 2012.

    Article  Google Scholar 

  26. Kwon, R. Y., D. R. Meays, W. J. Tang, and J. A. Frangos. Microfluidic enhancement of intramedullary pressure increases interstitial fluid flow and inhibits bone loss in hindlimb suspended mice. J. Bone Miner. Res. 25:1798–1807, 2010.

    Article  Google Scholar 

  27. Kwon, R. Y., S. Temiyasathit, P. Tummala, C. C. Quah, and C. R. Jacobs. Primary cilium-dependent mechanosensing is mediated by adenylyl cyclase 6 and cyclic AMP in bone cells. FASEB J. 24:2859–2868, 2010.

    Article  Google Scholar 

  28. Li, Z., Z. Wang, L. Yang, X. Li, Y. Sasaki, S. Wang, S. Araki, M. Mezawa, H. Takai, Y. Nakayama, and Y. Ogata. Fibroblast growth factor 2 regulates bone sialoprotein gene transcription in human breast cancer cells. J. Oral Sci. 52:125–132, 2010.

    Article  Google Scholar 

  29. Liu, J., M. W. Albers, T. J. Wandless, S. Luan, D. G. Alberg, P. J. Belshaw, P. Cohen, C. MacKintosh, C. B. Klee, and S. L. Schreiber. Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry 31:3896–3901, 1992.

    Article  Google Scholar 

  30. Macian, F., C. Lopez-Rodriguez, and A. Rao. Partners in transcription: NFAT and AP-1. Oncogene 20:2476–2489, 2001.

    Article  Google Scholar 

  31. Malone, A. M., N. N. Batra, G. Shivaram, R. Y. Kwon, L. You, C. H. Kim, J. Rodriguez, K. Jair, and C. R. Jacobs. The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts. Am. J. Physiol. Cell Physiol. 292:C1830–C1836, 2007.

    Article  Google Scholar 

  32. McCabe, L. R., C. Banerjee, R. Kundu, R. J. Harrison, P. R. Dobner, J. L. Stein, J. B. Lian, and G. S. Stein. Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation. Endocrinology 137:4398–4408, 1996.

    Google Scholar 

  33. McCabe, L. R., M. Kockx, J. Lian, J. Stein, and G. Stein. Selective expression of fos- and jun-related genes during osteoblast proliferation and differentiation. Exp. Cell Res. 218:255–262, 1995.

    Article  Google Scholar 

  34. Orcel, P., M. A. Denne, and M. C. de Vernejoul. Cyclosporin-A in vitro decreases bone resorption, osteoclast formation, and the fusion of cells of the monocyte–macrophage lineage. Endocrinology 128:1638–1646, 1991.

    Article  Google Scholar 

  35. Palkowitsch, L., U. Marienfeld, C. Brunner, A. Eitelhuber, D. Krappmann, and R. B. Marienfeld. The Ca2+-dependent phosphatase calcineurin controls the formation of the Carma1-Bcl10-Malt1 complex during T cell receptor-induced NF-kappaB activation. J. Biol. Chem. 286:7522–7534, 2011.

    Article  Google Scholar 

  36. Peverali, F. A., E. K. Basdra, and A. G. Papavassiliou. Stretch-mediated activation of selective MAPK subtypes and potentiation of AP-1 binding in human osteoblastic cells. Mol. Med. 7:68–78, 2001.

    Google Scholar 

  37. Pfaffl, M. W., G. W. Horgan, and L. Dempfle. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30:e36, 2002.

    Article  Google Scholar 

  38. Pruitt, L. A., D. R. Taaffe, and R. Marcus. Effects of a one-year high-intensity versus low-intensity resistance training program on bone mineral density in older women. J. Bone Miner. Res. 10:1788–1795, 1995.

    Article  Google Scholar 

  39. Pyrzynska, B., G. Mosieniak, and B. Kaminska. Changes of the trans-activating potential of AP-1 transcription factor during cyclosporin A-induced apoptosis of glioma cells are mediated by phosphorylation and alterations of AP-1 composition. J. Neurochem. 74:42–51, 2000.

    Article  Google Scholar 

  40. Qiu, N., Z. Xiao, L. Cao, M. M. Buechel, V. David, E. Roan, and L. D. Quarles. Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia. J. Cell Sci. 125:1945–1957, 2012.

    Article  Google Scholar 

  41. Ramakers, C., J. M. Ruijter, R. H. Deprez, and A. F. Moorman. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339:62–66, 2003.

    Article  Google Scholar 

  42. Rao, A., C. Luo, and P. G. Hogan. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15:707–747, 1997.

    Article  Google Scholar 

  43. Rauch, C., and P. T. Loughna. Stretch-induced activation of ERK in myocytes is p38 and calcineurin-dependent. Cell Biochem. Funct. 26:866–869, 2008.

    Article  Google Scholar 

  44. Rubin, C., A. S. Turner, S. Bain, C. Mallinckrodt, and K. McLeod. Anabolism. Low mechanical signals strengthen long bones. Nature 412:603–604, 2001.

    Article  Google Scholar 

  45. Schaefer, A., M. Magocsi, A. Fandrich, and H. Marquardt. Stimulation of the Ca2+-mediated egr-1 and c-fos expression in murine erythroleukemia cells by cyclosporin A. Biochem. J. 335(Pt 3):505–511, 1998.

    Google Scholar 

  46. Srinivasan, S., B. J. Ausk, J. Prasad, D. Threet, S. D. Bain, T. S. Richardson, and T. S. Gross. Rescuing loading induced bone formation at senescence. PLoS Comput. Biol. 6:e1000924, 2010.

    Article  Google Scholar 

  47. Srinivasan, S., T. S. Gross, and S. D. Bain. Bone mechanotransduction may require augmentation in order to strengthen the senescent skeleton. Ageing Res. Rev. 11:353–360, 2012.

    Article  Google Scholar 

  48. Srinivasan, S., D. Threet, L. E. Worton, B. J. Ausk, S. D. Bain, E. M. Gardiner, R. Y. Kwon, and T. S. Gross. Distinct cyclosporin a doses are required to enhance bone formation induced by cyclic and rest-inserted loading in the senescent skeleton. PLoS ONE 9:e84868, 2014.

    Article  Google Scholar 

  49. Sugimoto, T., S. Stewart, and K. L. Guan. The calcium/calmodulin-dependent protein phosphatase calcineurin is the major Elk-1 phosphatase. J. Biol. Chem. 272:29415–29418, 1997.

    Article  Google Scholar 

  50. Tamler, R., and S. Epstein. Nonsteroid immune modulators and bone disease. Ann. N Y Acad. Sci. 284–296:2006, 1068.

    Google Scholar 

  51. Worton, L. E., B. J. Ausk, L. M. Downey, S. D. Bain, E. M. Gardiner, S. Srinivasan, T. S. Gross, and R. Y. Kwon. Systems-based identification of temporal processing pathways during bone cell mechanotransduction. PLoS ONE 8(9):e74205, 2013.

    Article  Google Scholar 

  52. Wu, C. C., Y. S. Li, J. H. Haga, N. Wang, I. Y. Lian, F. C. Su, S. Usami, and S. Chien. Roles of MAP kinases in the regulation of bone matrix gene expressions in human osteoblasts by oscillatory fluid flow. J. Cell. Biochem. 98:632–641, 2006.

    Article  Google Scholar 

  53. Yeo, H., L. H. Beck, J. M. McDonald, and M. Zayzafoon. Cyclosporin A elicits dose-dependent biphasic effects on osteoblast differentiation and bone formation. Bone 40:1502–1516, 2007.

    Article  Google Scholar 

  54. Yeo, H., J. M. McDonald, and M. Zayzafoon. NFATc1: a novel anabolic therapeutic target for osteoporosis. Ann. N Y Acad. Sci. 564–567:2006, 1068.

    Google Scholar 

  55. Young, S. R., R. Gerard-O’Riley, M. Harrington, and F. M. Pavalko. Activation of NF-kappaB by fluid shear stress, but not TNF-alpha, requires focal adhesion kinase in osteoblasts. Bone 47:74–82, 2010.

    Article  Google Scholar 

  56. Young, S. R., J. M. Hum, E. Rodenberg, C. H. Turner, and F. M. Pavalko. Non-overlapping functions for Pyk2 and FAK in osteoblasts during fluid shear stress-induced mechanotransduction. PLoS ONE 6:e16026, 2011.

    Article  Google Scholar 

  57. Yu, S., Q. Geng, F. Sun, Y. Yu, Q. Pan, and A. Hong. Osteogenic differentiation of C2C12 myogenic progenitor cells requires the Fos-related antigen Fra-1—a novel target of Runx2. Biochem. Biophys. Res. Commun. 430:173–178, 2013.

    Article  Google Scholar 

  58. Zhou, S., E. M. Bueno, S. W. Kim, I. Amato, L. Shen, J. Hahne, I. Bleiberg, P. Morley, and J. Glowacki. Effects of age on parathyroid hormone signaling in human marrow stromal cells. Aging Cell 10:780–788, 2011.

    Article  Google Scholar 

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Acknowledgments

The authors would like to acknowledge financial support from the University of Washington Department of Orthopaedics and Sports Medicine (RYK), the National Institutes of Health (SS: AR0562235, TSG: AR056652), and the Sigvard T. Hansen, Jr. Endowed Chair (TSG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest

Leah E. Worton, Ronald Y. Kwon, Edith M. Gardiner, Ted S. Gross, and Sundar Srinivasan declare that they have no conflicts of interest.

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No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.

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Authors and Affiliations

  1. Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, WA, USA

    Leah E. Worton, Ronald Y. Kwon, Edith M. Gardiner, Ted S. Gross & Sundar Srinivasan

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  1. Leah E. Worton
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  2. Ronald Y. Kwon
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  3. Edith M. Gardiner
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Correspondence to Leah E. Worton.

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Associate Editor Michael R. King oversaw the review of this article.

Leah E. Worton and Ronald Y. Kwon have contributed equally to this work.

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Worton, L.E., Kwon, R.Y., Gardiner, E.M. et al. Enhancement of Flow-Induced AP-1 Gene Expression by Cyclosporin A Requires NFAT-Independent Signaling in Bone Cells. Cel. Mol. Bioeng. 7, 254–265 (2014). https://doi.org/10.1007/s12195-014-0321-3

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