Alteration of Stress Fiber in Fibroblastic Reticular Cells via Lymphotoxin β Receptor Stimulation is Associated with Myosin

Lymphotoxin β 수용체를 통한 fibroblastic reticular cell의 stress fiber 변화와 myosin의 연관성

Kim, Min Hwan;Kim, Yeon Hee;Choi, Woobong;Lee, Jong-Hwan

  • Received : 2015.02.21
  • Accepted : 2015.04.22
  • Published : 2015.05.30


Stress fiber (SF) alteration is mediated by cellular receptors, which, upon interaction with the extracellular counterpart, signal to the actin cytoskeleton for remodeling. This association is mediated by a variety of scaffold and signaling factors, which control the mechanical and signaling activities of the interaction site. The heterotrimeric transmembrane lymphotoxin α1β2 (LTα1β2), a member of the tumor necrosis factor (TNF) family of cytokines, including soluble homotrimeric lymphotoxin (LT α), plays an important role in lymphoid tissue architecture. Ligation between LTα1β2 and the lymphotoxin β receptor (LTβR) activates signal-cascade in fibroblastic reticular cells (FRCs). We found LTβR stimulation using an agonistic anti-LTβR antibody alone or combined with LTα or TNFα induced changes in the actin and plasticity of cells. To clarify the involvement of myosin underlying the alteration, we analyzed the effect of myosin light chain kinase (MLCK) with an MLCK inhibitor (ML7), the phosphorylation level of myosin light chains (MLC), and the level of phospho-myosin phosphatase target subunit 1 (MYPT1) after treatment with an agonistic anti-LTβR antibody for cytoskeleton reorganization in FRCs. The inhibition of MLCK activity induced changes in the actin cytoskeleton organization and cell morphology in FRC. In addition, we showed the phosphorylation of MLC and MYPT1 was reduced by LTβR stimulation in cells. A DNA chip revealed the LTβR stimulation of FRC down-regulated transcripts of myosin and actin components. Collectively, these results suggest LTβR stimulation is linked to myosin regarding SF alteration in FRC.


Fibroblastic reticular cell (FRC);lymphotoxin β;receptor (LTβR);myosin;stress fiber


  1. Sudhamsu, J., Yin, J., Chiang, E. Y., Starovasnik, M. A., Grogan, J. L. and Hymowitz, S. G. 2013. Dimerization of LTβR by LTα1β2 is necessary and sufficient for signal transduction. Proc. Natl. Acad. Sci. USA 110, 19896-19901.
  2. Rumbo, M., Sierro, F., Debard, N., Kraehenbuhl, J. P. and Finke, D. 2004. Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium. Gastroenterology 127, 213-223.
  3. Šedý, J., Bekiaris, V. and Ware, C. F. 2014. Tumor Necrosis Factor Superfamily in Innate Immunity and Inflammation. Cold Spring Harb. Perspect. Biol. 7, a016279.
  4. Sobocinski, G. P., Toy, K., Bobrowski, W. F., Shaw, S., Anderson, A. O. and Kaldjian, E. P. 2010. Ultrastructural localization of extracellular matrix proteins of the lymphnode cortex: evidence supporting the reticular network as a pathway for lymphocyte migration. BMC Immunol. doi:10.1186/1471-2172-11-42.
  5. Spindel, O. N., Burke, R. M., Yan, C. and Berk, B. C. 2014. Thioredoxin-interacting protein is a biomechanical regulator of Src activity: key role in endothelial cell SF formation. Circ. Res. 114, 1125-1132.
  6. Stachowiak, M. R., Smith, M. A., Blankman, E., Chapin, L. M., Balcioglu, H. E., Wang, S., Beckerle, M. C. and O'Shaughnessy, B. 2014. A mechanical-biochemical feed backloop regulates remodeling in the actin cytoskeleton. Proc. Natl. Acad. Sci. USA 111, 17528-17533.
  7. Tripathi, B. K., Lowy, D. R. and Zelenka, P. S. 2015. The Cdk5 activator P39 specifically links muskelin to myosin II and regulates SF formation and actin organization in lens. Exp. Cell Res. 330, 186-198.
  8. Zhu, M. and Fu, Y. X. 2011. The role of core TNF/LIGHT family members in lymph node homeostasis and remodeling. Immunol. Rev. 244, 75-84.
  9. Kelleher, Z. T., Sha, Y., Foster, M. W., Foster, W. M., Forrester, M. T. and Marshall, H. E. 2014. Thioredoxin-mediated denitrosylation regulates cytokine-induced nuclear factor κB (Marshall, H. ENF-κB) activation. J. Biol. Chem. 289, 3066-3072.
  10. Nie, W., Wei, M. T., Ou-Yang, H. D., Jedlicka, S. S. and Vavylonis, D. 2015. Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover,contraction, and stress-stabilization. Cytoskeleton (Hoboken).doi: 10.1002/cm.21207.
  11. Korczyński, J., Sobierajska, K., Krzemiński, P., Wasik, A., Wypych, D., Pomorski, P. and Kłopocka, W. 2011. Is MLC phosphorylation essential for the recovery from ROCK inhibition in glioma C6 cells? Acta. Biochim. Pol. 58, 125-130.
  12. Lo, J. C., Basak, S., James, E. S., Quiambo, R. S., Kinsella, M. C., Alegre, M. L., Weih, F., Franzoso, G., Hoffmann, A. and Fu, Y. X. 2006. Coordination between NF-kappaB family members p50 and p52 is essential for mediating LTbetaR signals in the development and organization of secondary lymphoid tissues. Blood 107, 1048-1055.
  13. Michaelson, J. S., Demarest, S. J., Miller, B., Amatucci, A., Snyder, W. B., Wu, X., Huang, F., Phan, S., Gao, S., Doern, A., Farrington, G. K., Lugovskoy, A., Joseph, I., Bailly, V., Wang, X., Garber, E., Browning, J. and Glaser, S. M. 2009. Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR. MAbs 1, 128-141.
  14. Pellegrin, S. and Mellor, H. 2007. Actin stress fibres. J. Cell Sci. 120, 3491-3499.
  15. Qiao, Y. N., He, W. Q., Chen, C. P., Zhang, C. H., Zhao, W., Wang, P., Zhang, L., Wu, Y. Z., Yang, X., Peng, Y. J., Gao, J. M., Kamm, K. E., Stull, J. T. and Zhu, M. S. 2014. Myosin phosphatase target subunit 1 (MYPT1) regulates the contraction and relaxation of vascular smooth muscle and maintains blood pressure. J. Biol. Chem. 289, 22512-22523.
  16. Calmon-Hamaty, F., Combe, B., Hahne, M. and Morel, J. 2011. Lymphotoxin α stimulates proliferation and pro-inflammatory cytokine secretion of rheumatoid arthritis synovial fibroblasts. Cytokine 53, 207-214.
  17. Astarita, J. L., Cremasco, V., Fu, J., Darnell, M. C., Peck, J. R., Nieves-Bonilla, J. M., Song, K., Kondo, Y., Woodruff, M. C., Gogineni, A., Onder, L., Ludewig, B., Weimer, R. M., Carroll, M. C., Mooney, D. J., Xia, L. and Turley, S. J. 2015. The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture. Nat. Immunol. 16, 75-84.
  18. Berrier, A. L. and Yamada, K. M. 2007. Cell-matrix adhesion. J. Cell Physiol. 213, 565-573.
  19. Brown, F. D. and Turley, S. J. 2015. Fibroblastic reticular cells: organization and regulation of the T lymphocyte life cycle. J. Immunol. 194, 1389-1394.
  20. Chen, C., Tao, T., Wen, C., He, W. Q., Qiao, Y. N., Gao, Y. Q., Chen, X., Wang, P., Chen, C. P., Zhao, W., Chen, H. Q., Ye, A. P., Peng, Y. J. and Zhu, M. S. 2014. Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism. J. Biol. Chem. 289, 28478-28488.
  21. De Franceschi, N., Hamidi, H., Alanko, J., Sahgal, P. and Ivaska, J. 2015. Integrin traffic - the update. J. Cell Sci. pii: jcs.161653.
  22. Hirata, H., Gupta, M., Vedula, S. R., Lim, C. T., Ladoux, B. and Sokabe, M. 2015. Actomyosin bundles serve as a tension sensor and a platform for ERK activation. EMBO Rep. 16, 250-257.
  23. Katakai, T., Hara, T., Sugai, M., Gonda, H. and Shimizu, A. 2004. Lymph node fibroblastic reticular cells construct the stromal reticulum via contact with lymphocytes. J. Exp. Med. 200, 783-795.
  24. Acton, S. E., Farrugia, A. J., Astarita, J. L., Mourão-Sá, D., Jenkins, R. P., Nye, E., Hooper, S., van Blijswijk, J., Rogers, N. C., Snelgrove, K. J., Rosewell, I., Moita, L. F., Stamp, G., Turley, S. J., Sahai, E. and Reis e Sousa, C. Dendritic cells control fibroblastic reticular network tension and lymph node expansion. Nature 514, 498-502.