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Annals of Biomedical Engineering

, Volume 46, Issue 1, pp 48–59 | Cite as

Low-Intensity Ultrasound Modulates Ca2+ Dynamics in Human Mesenchymal Stem Cells via Connexin 43 Hemichannel

  • Chi Woo Yoon
  • Hayong Jung
  • Kyosuk Goo
  • Sunho Moon
  • Kweon Mo Koo
  • Nan Sook Lee
  • Andrew C. Weitz
  • K. Kirk ShungEmail author
Article

Abstract

In recent years, ultrasound has gained attention in new biological applications due to its ability to induce specific biological responses at the cellular level. Although the biophysical mechanisms underlying the interaction between ultrasound and cells are not fully understood, many agree on a pivotal role of Ca2+ signaling through mechanotransduction pathways. Because Ca2+ regulates a vast range of downstream cellular processes, a better understanding of how ultrasound influences Ca2+ signaling could lead to new applications for ultrasound. In this study, we investigated the mechanism of ultrasound-induced Ca2+ mobilization in human mesenchymal stem cells using 47 MHz focused ultrasound to stimulate single cells at low intensities (~ 110 mW/cm2). We found that ultrasound exposure triggers opening of connexin 43 hemichannels on the plasma membrane, causing release of ATP into the extracellular space. That ATP then binds to G-protein-coupled P2Y1 purinergic receptors on the membrane, in turn activating phospholipase C, which evokes production of inositol trisphosphate and release of Ca2+ from intracellular stores.

Keywords

Calcium signaling Mechanotransduction Purinergic signaling ATP 

Notes

Acknowledgments

This work was supported by the National Institutes of Health under Grant No. P41-EB002182 to Dr. K. Kirk Shung. We thank Hae Gyun Lim and Nestor Cabrera Muñoz for their help on the transducer fabrication. We thank Madison Zitting for proofreading.

Supplementary material

10439_2017_1949_MOESM1_ESM.docx (12 kb)
Supplementary material 1 (DOCX 12 kb)
10439_2017_1949_MOESM2_ESM.avi (1.6 mb)
Supplementary material 2 (AVI 1600 kb)
10439_2017_1949_MOESM3_ESM.avi (843 kb)
Supplementary material 3 (AVI 843 kb)

References

  1. 1.
    Alvarenga, E. C., R. Rodrigues, A. Caricati-Neto, F. C. Silva-Filho, E. J. Paredes-Gamero, and A. T. Ferreira. Low-intensity pulsed ultrasound-dependent osteoblast proliferation occurs by via activation of the P2Y receptor: role of the P2Y1 receptor. Bone 46:355–362, 2010.CrossRefPubMedGoogle Scholar
  2. 2.
    Arcuino, G., J. H. Lin, T. Takano, C. Liu, L. Jiang, Q. Gao, J. Kang, and M. Nedergaard. Intercellular calcium signaling mediated by point-source burst release of ATP. Proc. Natl Acad. Sci. USA 99:9840–9845, 2002.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bao, L., F. Sachs, and G. Dahl. Connexins are mechanosensitive. Amer. J. Physiol. Cell Physiol. 287:C1389–C1395, 2004.CrossRefGoogle Scholar
  4. 4.
    Batra, N., S. Burra, A. J. Siller-Jackson, S. Gu, X. Xia, G. F. Weber, D. DeSimone, L. F. Bonewald, E. M. Lafer, E. Sprague, M. A. Schwartz, and J. X. Jiang. Mechanical stress-activated integrin α5β1 induces opening of connexin 43 hemichannels. Proc. Natl Acad. Sci. 109:3359–3364, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Berridge, M. J. The AM and FM of calcium signalling. Nature 386:759–760, 1997.CrossRefPubMedGoogle Scholar
  6. 6.
    Berridge, M. J. Cell signalling. A tale of two messengers. Nature 365:388–389, 1993.CrossRefPubMedGoogle Scholar
  7. 7.
    Berridge, M. J. Dysregulation of neural calcium signaling in Alzheimer disease, bipolar disorder and schizophrenia. Prion 7:2–13, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Delaine-Smith, R. M., and G. C. Reilly. Mesenchymal stem cell responses to mechanical stimuli. Muscles Ligaments Tendons J 2:169–180, 2012.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Etienne-Manneville, S., J. B. Manneville, P. Adamson, B. Wilbourn, J. Greenwood, and P. O. Couraud. ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines. J Immunol 165:3375–3383, 2000.CrossRefPubMedGoogle Scholar
  10. 10.
    Fan J., Z. H. Lee, W. C. Ng, W. L. Khoa, S. H. Teoh, T. H. Soong, Y. R. Qin, Z. Y. Zhang and X. P. Li. Effect of pulse magnetic field stimulation on calcium channel current. Fifth Moscow international symposium on magnetism 324: 3491–3494, 2012Google Scholar
  11. 11.
    Fan, Z., R. E. Kumon, J. Park, and C. X. Deng. Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles. J Control Release 142:31–39, 2010.CrossRefPubMedGoogle Scholar
  12. 12.
    Garcia, M., and M. M. Knight. Cyclic loading opens hemichannels to release ATP as part of a chondrocyte mechanotransduction pathway. J Orthop Res 28:510–515, 2010.PubMedGoogle Scholar
  13. 13.
    Ghosh, A., and M. E. Greenberg. Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268:239–247, 1995.CrossRefPubMedGoogle Scholar
  14. 14.
    Goñi, G. M., C. Epifano, J. Boskovic, M. Camacho-Artacho, J. Zhou, A. Bronowska, M. T. Martín, M. J. Eck, L. Kremer, F. Gräter, F. L. Gervasio, M. Perez-Moreno, and D. Lietha. Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes. Proc. Natl Acad. Sci. 111:E3177–E3186, 2014.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Huang, J., Z. Ye, X. Hu, L. Lu, and Z. Luo. Electrical stimulation induces calcium-dependent release of NGF from cultured Schwann cells. Glia 58:622–631, 2010.PubMedGoogle Scholar
  16. 16.
    Hwang, J. Y., N. S. Lee, C. Lee, K. H. Lam, H. H. Kim, J. Woo, M. Y. Lin, K. Kisler, H. Choi, Q. Zhou, R. H. Chow, and K. K. Shung. Investigating contactless high frequency ultrasound microbeam stimulation for determination of invasion potential of breast cancer cells. Biotechnol Bioeng 110:2697–2705, 2013.CrossRefPubMedGoogle Scholar
  17. 17.
    Hwang, J. Y., H. G. Lim, C. W. Yoon, K. H. Lam, S. Yoon, C. Lee, C. T. Chiu, B. J. Kang, H. H. Kim, and K. K. Shung. Non-contact high-frequency ultrasound microbeam stimulation for studying mechanotransduction in human umbilical vein endothelial cells. Ultrasound Med Biol 40:2172–2182, 2014.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Izquierdo-Serra, M., D. Trauner, A. Llobet, and P. Gorostiza. Optical modulation of neurotransmission using calcium photocurrents through the ion channel LiGluR. Front Mol Neurosci 6:3, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Jiang, J. X., A. J. Siller-Jackson, and S. Burra. Roles of gap junctions and hemichannels in bone cell functions and in signal transmission of mechanical stress. Front. Biosci. J. Virtual Libr. 12:1450–1462, 2007.CrossRefGoogle Scholar
  20. 20.
    Kang, K. S., J. M. Hong, J. A. Kang, J. W. Rhie, and D. W. Cho. Osteogenic differentiation of human adipose-derived stem cells can be accelerated by controlling the frequency of continuous ultrasound. J. Ultrasound Med. 32:1461–1470, 2013.CrossRefPubMedGoogle Scholar
  21. 21.
    Kim, T. J., C. Joo, J. Seong, R. Vafabakhsh, E. L. Botvinick, M. W. Berns, A. E. Palmer, N. Wang, T. Ha, E. Jakobsson, J. Sun, and Y. Wang. Distinct mechanisms regulating mechanical force-induced Ca2+ signals at the plasma membrane and the ER in human MSCs. Elife 4:e04876, 2015.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Kim, T. J., J. Sun, S. Lu, Y. X. Qi, and Y. Wang. Prolonged mechanical stretch initiates intracellular calcium oscillations in human mesenchymal stem cells. PLoS ONE 9:e109378, 2014.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Kimmel, E. Cavitation bioeffects. Crit. Rev. Biomed. Eng. 34:105–161, 2006.CrossRefPubMedGoogle Scholar
  24. 24.
    Krasovitski, B., V. Frenkel, S. Shoham, and E. Kimmel. Intramembrane cavitation as a unifying mechanism for ultrasound-induced bioeffects. Proc. Natl Acad. Sci. USA 108:3258–3263, 2011.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kumon, R. E., M. Aehle, D. Sabens, P. Parikh, Y. W. Han, D. Kourennyi, and C. X. Deng. Spatiotemporal effects of sonoporation measured by real-time calcium imaging. Ultrasound Med. Biol. 35:494–506, 2009.CrossRefPubMedGoogle Scholar
  26. 26.
    Kusuyama, J., K. Bandow, M. Shamoto, K. Kakimoto, T. Ohnishi, and T. Matsuguchi. Low intensity pulsed ultrasound (LIPUS) influences the multilineage differentiation of mesenchymal stem and progenitor cell lines through ROCK-Cot/Tpl2-MEK-ERK signaling pathway. J. Biol. Chem. 289:10330–10344, 2014.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lam, K. H., H. S. Hsu, Y. Li, C. Lee, A. Lin, Q. Zhou, E. S. Kim, and K. K. Shung. Ultrahigh frequency lensless ultrasonic transducers for acoustic tweezers application. Biotechnol. Bioeng. 110:881–886, 2013.CrossRefPubMedGoogle Scholar
  28. 28.
    Lee, H. J., B. H. Choi, B. H. Min, Y. S. Son, and S. R. Park. Low-intensity ultrasound stimulation enhances chondrogenic differentiation in alginate culture of mesenchymal stem cells. Artif. Org. 30:707–715, 2006.CrossRefGoogle Scholar
  29. 29.
    Leybaert, L., and M. J. Sanderson. Intercellular Ca(2+) waves: mechanisms and function. Physiological Reviews 92:1359–1392, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Liu, Y. S., Y. A. Liu, C. J. Huang, M. H. Yen, C. T. Tseng, S. Chien, and O. K. Lee. Mechanosensitive TRPM7 mediates shear stress and modulates osteogenic differentiation of mesenchymal stromal cells through Osterix pathway. Sci. Rep. 5:16522, 2015.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Missiaen, L., W. Robberecht, L. van den Bosch, G. Callewaert, J. B. Parys, F. Wuytack, L. Raeymaekers, B. Nilius, J. Eggermont, and H. De Smedt. Abnormal intracellular Ca(2+)homeostasis and disease. Cell Calcium 28:1–21, 2000.CrossRefPubMedGoogle Scholar
  32. 32.
    Morgado-Valle, C., L. Verdugo-Díaz, D. E. García, C. Morales-Orozco, and R. Drucker-Colín. The role of voltage-gated Ca2+ channels in neurite growth of cultured chromaffin cells induced by extremely low frequency (ELF) magnetic field stimulation. Cell Tissue Res. 291:217–230, 1998.CrossRefPubMedGoogle Scholar
  33. 33.
    Nelson, T. R., J. B. Fowlkes, J. S. Abramowicz, and C. C. Church. Ultrasound biosafety considerations for the practicing sonographer and sonologist. J. Ultrasound Med. 28:139–150, 2009.CrossRefPubMedGoogle Scholar
  34. 34.
    Orr, A. W., B. P. Helmke, B. R. Blackman, and M. A. Schwartz. Mechanisms of mechanotransduction. Dev. Cell 10:11–20, 2006.CrossRefPubMedGoogle Scholar
  35. 35.
    Padilla, F., R. Puts, L. Vico, and K. Raum. Stimulation of bone repair with ultrasound: a review of the possible mechanic effects. Ultrasonics 54:1125–1145, 2014.CrossRefPubMedGoogle Scholar
  36. 36.
    Parvizi, J., V. Parpura, J. F. Greenleaf, and M. E. Bolander. Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes. J. Orthop. Res. 20:51–57, 2002.CrossRefPubMedGoogle Scholar
  37. 37.
    Pessina, G. P., C. Aldinucci, M. Palmi, G. Sgaragli, A. Benocci, A. Meini, and F. Pessina. Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells. Bioelectromagnetics 22:503–510, 2001.CrossRefPubMedGoogle Scholar
  38. 38.
    Plaksin, M., S. Shoham, and E. Kimmel. Intramembrane cavitation as a predictive bio-piezoelectric mechanism for ultrasonic brain stimulation. Phys. Rev. X 4:011004, 2014.Google Scholar
  39. 39.
    Pounder, N. M., and A. J. Harrison. Low intensity pulsed ultrasound for fracture healing: a review of the clinical evidence and the associated biological mechanism of action. Ultrasonics 48:330–338, 2008.CrossRefPubMedGoogle Scholar
  40. 40.
    Roderick, H. L., and S. J. Cook. Ca2 + signalling checkpoints in cancer: remodelling Ca2+ for cancer cell proliferation and survival. Nat. Rev. Cancer 8:361–375, 2008.CrossRefPubMedGoogle Scholar
  41. 41.
    Sanderson, M. J., A. C. Charles, S. Boitano, and E. R. Dirksen. Mechanisms and function of intercellular calcium signaling. Mol. Cell Endocrinol 98:173–187, 1994.CrossRefPubMedGoogle Scholar
  42. 42.
    Simone, L. C., S. Caplan, and N. Naslavsky. Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization. PLoS ONE 8:e74519, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Takada, H., K. Furuya, and M. Sokabe. Mechanosensitive ATP release from hemichannels and Ca2+ influx through TRPC6 accelerate wound closure in keratinocytes. J. Cell Sci. 127:4159–4171, 2014.CrossRefPubMedGoogle Scholar
  44. 44.
    Tan, T., J. Xie, Z. Tong, T. Liu, X. Chen, and X. Tian. Repetitive transcranial magnetic stimulation increases excitability of hippocampal CA1 pyramidal neurons. Brain Res. 1520:23–35, 2013.CrossRefPubMedGoogle Scholar
  45. 45.
    Thi, M. M., S. Islam, S. O. Suadicani, and D. C. Spray. Connexin43 and Pannexin1 Channels in Osteoblasts: Who Is the “Hemichannel”? J. Membr. Biol. 245:401–409, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Weitz, A. C., N. S. Lee, C. W. Yoon, A. Bonyad, K. S. Goo, S. Kim, S. Moon, H. Jung, Q. Zhou, R. H. Chow, and K. K. Shung. Functional assay of cancer cell invasion potential based on mechanotransduction of focused ultrasound. Front. Oncol. 7:161, 2017.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Xiao, E., C. Chen, and Y. Zhang. The mechanosensor of mesenchymal stem cells: mechanosensitive channel or cytoskeleton? Stem Cell Res. Ther 7:140, 2016.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Xiao, E., H. Q. Yang, Y. H. Gan, D. H. Duan, L. H. He, Y. Guo, S. Q. Wang, and Y. Zhang. Brief reports: TRPM7 Senses mechanical stimulation inducing osteogenesis in human bone marrow mesenchymal stem cells. Stem Cells 33:615–621, 2015.CrossRefPubMedGoogle Scholar
  49. 49.
    Yoon, J. H., E. Y. Roh, S. Shin, N. H. Jung, E. Y. Song, D. S. Lee, K. S. Han, J. S. Kim, B. J. Kim, H. W. Jeon, and K. S. Yoon. Introducing pulsed low-intensity ultrasound to culturing human umbilical cord-derived mesenchymal stem cells. Biotechnol. Lett. 31:329–335, 2009.CrossRefPubMedGoogle Scholar

Copyright information

© Biomedical Engineering Society 2017

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringUniversity of Southern CaliforniaLos AngelesUSA
  2. 2.Institute for Biomedical TherapeuticsUniversity of Southern CaliforniaLos AngelesUSA

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