Extracellular matrix cues modulate Schwann cell morphology, proliferation, and protein expression
Zhenyuan Xu
Department of Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio
Search for more papers by this authorJacob A. Orkwis
Department of Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio
Search for more papers by this authorBraden M. DeVine
Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio
Search for more papers by this authorCorresponding Author
Greg M. Harris
Department of Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio
Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio
Correspondence
Greg Harris, PhD, Department of Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, OH 45221.
Email: [email protected]
Search for more papers by this authorZhenyuan Xu
Department of Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio
Search for more papers by this authorJacob A. Orkwis
Department of Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio
Search for more papers by this authorBraden M. DeVine
Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio
Search for more papers by this authorCorresponding Author
Greg M. Harris
Department of Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio
Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio
Correspondence
Greg Harris, PhD, Department of Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, OH 45221.
Email: [email protected]
Search for more papers by this authorAbstract
Peripheral nerve injuries require a complex set of signals from cells, macrophages, and the extracellular matrix (ECM) to induce regeneration across injury sites and achieve functional recovery. Schwann cells (SCs), the major glial cell in the peripheral nervous system (PNS), are critical to nerve regeneration due to their inherent capacity for altering phenotype postinjury to facilitate wound healing. The ECM plays a vital role in wound healing as well as regulating cell phenotype during tissue repair. To examine the underlying mechanisms between the ECM and SCs, this work sought to determine how specific ECM cues regulate the phenotype of SCs. To address this, SCs were cultured on polydimethylsiloxane substrates of a variable Young's modulus coated with ECM proteins. Cells were analyzed for spreading area, proliferation, cell and nuclear shape, and c-Jun expression. It was found that substrates with a stiffness of 8.67 kPa coated with laminin promoted the highest expression of c-Jun, a marker signifying a “regenerative” SC. Microcontact printed, cell adhesive areas were then utilized to precisely control the geometry and spreading of SCs and by controlling spreading area and cellular elongation; expression of c-Jun was either promoted or downregulated. These results begin to address the significant interplay between ECM cues and phenotype of SCs, while offering a potential means to enhance PNS regeneration through cellular therapies.
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.
Supporting Information
| Filename | Description |
|---|---|
| term2987-sup-0001-Supplementary_Table.docxWord 2007 document , 14.4 KB | Table S1: Table of statistical significance showing c-Jun fluorescent intensity between different substrate conditions. |
| term2987-sup-0002-Supplementary_S1-S9.pdfPDF document, 1.6 MB |
Figure S1: RT4-D6P2T cells immunolabeled with S100 and DAPI to test the purity of Schwann cell. Figure S2: Histogram showing the Young's moduli as the mixing ratio of PDMS base to curing agent. Figure S3: Histograms showing spreading area of SC sseeded on (A) Uncoated PDMS (B) Collagen I coated PDMS (C) Fibronectin coated PDMS and (D) Laminin coated PDMS respectively. Figure S4: Histograms showing the percentage of BrdU incorporation of SCs seeded on (A) Uncoated PDMS (B) Collagen I coated PDMS (C) Fibronectin coated PDMS and (D) Laminin coated PDMS respectively. Figure S5: Histograms showing the fluorescent intensity of c-Jun in SCs cultured on (A) Uncoated PDMS (B) Collagen I coated PDMS (C) Fibronectin coated PDMS and (D) Laminin coated PDMS respectively. Figure S6: Western blot quantification of (A) c-Jun and (B) MBP expression in SCs. Figure S7: Histograms showing the nuclear aspect ratio of SCs seeded on (A) Uncoated PDMS (B) Collagen I coated PDMS (C) Fibronectin coated PDMS and (D) Laminin coated PDMS respectively. Figure S8: The average c-Jun intensity was promoted with a higher nuclear aspect ratio in SCs. The nuclear aspect ratio of individual SCs seeded on protein coated PDMS was classified into four separate categories.The mean pixel intensity of c-Jun corresponding to each category shown with collagen I, fibronectin, and laminin histograms. Figure S9: c-Jun mean pixel intensity was related to cell spreading area in SCs. SC spreading area of individual cells cultured on substrates was classified into four distinct categories with the mean pixel intensity of c-Jun shown in the histograms. The mean pixel intensity of c-Jun corresponding to each category is shown in the histogram with collagen I, fibronectin and laminin coated surfaces. |
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REFERENCES
- Arthur, W. T., Burridge, K., Carolina, N., Hill, C., & Carolina, N. (2001). RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity. Molecular Biology of the Cell, 12, 2711–2720. https://doi.org/10.1091/mbc.12.9.2711
- Arthur-Farraj, P. J., Latouche, M., Wilton, D. K., Quintes, S., Chabrol, E., Banerjee, A., … Jessen, K. R. (2012). c-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for regeneration. Neuron, 75, 633–647. https://doi.org/10.1016/j.neuron.2012.06.021
- Baron-Van Evercooren, A., Gansmuller, A., Gumpel, M., Baumann, N., & Kleinman, H. K. (1986). Schwann cell differentiation in vitro: Extracellular matrix deposition and interaction. Developmental Neuroscience, 8(3), 182–196. https://doi.org/10.1159/000112252
- Chernousov, M. A., & Carey, D. J. (2000). Schwann cell extracellular matrix molecules and their receptors. Histology and Histopathology, 15, 593–601. https://doi.org/10.14670/HH-15.593
- Chernousov, M. A., Yu, W.-M., Chen, Z.-L., Carey, D. J., & Strickland, S. (2008). Regulation of Schwann cell function by the extracellular matrix. Glia, 56, 1498–1507. https://doi.org/10.1002/glia.20740
- Coso, O. A., Chiariello, M., & Yu, J. C. (1995). The small GTP-binding proteins Racl, and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell, 81, 1137–1146. https://doi.org/10.1016/s0092-8674(05)80018-2
- Cs, P., Pb, S., Mg, G., & Mjg, R. (2016). The role of the basal lamina in nerve regeneration. Journal of Cytology & Histology, 7, 4–8. https://doi.org/10.4172/2157-7099.1000438
10.4172/2157?7099.1000438 Google Scholar
- Deumens, R., Bozkurt, A., Meek, M. F., Marcus, M. A. E., Joosten, E. A. J., Weis, J., & Brook, G. A. (2010). Repairing injured peripheral nerves: Bridging the gap. Progress in Neurobiology, 92, 245–276. https://doi.org/10.1016/j.pneurobio.2010.10.002
- Dupont, S., Morsut, L., Aragona, M., Enzo, E., Giulitti, S., Cordenonsi, M., … Piccolo, S. (2011). Role of YAP/TAZ in mechanotransduction. Nature, 474, 179–184. https://doi.org/10.1038/nature10137
- Etienne-manneville, S. A. H. (2002). Rho GTPases in cell biology. Nature, 420, 629–635. https://doi.org/10.1038/nature01148
- Grove, M., Kim, H., Santerre, M., Krupka, A. J., Han, S. B., Zhai, J., … Son, Y. J. (2017). YAP/TAZ initiate and maintain schwann cell myelination. eLife, 6, 1–27. https://doi.org/10.7554/eLife.20982
- Gu, Y., Ji, Y., Zhao, Y., Liu, Y., Ding, F., Gu, X., & Yang, Y. (2012). The influence of substrate stiffness on the behavior and functions of Schwann cells in culture. Biomaterials, 33, 6672–6681. https://doi.org/10.1016/j.biomaterials.2012.06.006
- Gutekunst, S. B., Grabosch, C., Kovalev, A., Gorb, S. N., & Selhuber-unkel, C. (2014). Influence of the PDMS substrate stiffness on the adhesion of Acanthamoeba castellanii. Beilstein Journal of Nanotechnology, 5, 1393–1398. https://doi.org/10.3762/bjnano.5.152
- Harris, G. M., Madigan, N. N., Lancaster, K. Z., Enquist, L. W., Windebank, A. J., Schwartz, J., & Schwarzbauer, J. E. (2017). Nerve guidance by a decellularized fibroblast extracellular matrix. Matrix Biology, 60-61, 176–189. https://doi.org/10.1016/j.matbio.2016.08.011
- Harris, G. M., Piroli, M. E., & Jabbarzadeh, E. (2014). Deconstructing the effects of matrix elasticity and geometry in mesenchymal stem cell lineage commitment. Advanced Functional Materials, 24(16), 2396–2403. https://doi.org/10.1002/adfm.201303400
- Hoffman-Kim, D., Mitchel, J. A., & Bellamkonda, R. V. (2010). Topography, cell response, and nerve regeneration. Annual Review of Biomedical Engineering, 12, 203–231. https://doi.org/10.1146/annurev-bioeng-070909-105351
- Holmes, D. F., Lu, H., Richardson, S., Kadler, K. E., & Building, M. S. (2008). Ageing changes in the tensile properties of tendons: Influence of collagen fibril volume. Journal of Biomechanical Engineering, 130, 1–8. https://doi.org/10.1115/1.2898732
- Jessen, K. R., & Mirsky, R. (2016). The repair Schwann cell and its function in regenerating nerves. Journal of Physiology, 594, 3521–3531. https://doi.org/10.1113/JP270874
- Ju, M. S., Lin, C. C. K., & Chang, C. T. (2017). Researches on biomechanical properties and models of peripheral nerves—A review. Journal of Biomechanical Science and Engineering, 12, s. https://doi.org/10.1299/jbse.16-00678
10.1299/jbse.16?00678 Google Scholar
- Keung, A. J., De Juan-Pardo, E. M., Schaffer, D. V., & Kumar, S. (2011). Rho GTPases mediate the mechanosensitive lineage commitment of neural stem cells. Stem Cells, 29, 1886–1897. https://doi.org/10.1002/stem.746
- Khalili, A. A., & Ahmad, M. R. (2015). A review of cell adhesion studies for biomedical and biological applications. International Journal of Molecular Sciences, 16, 18149–18184. https://doi.org/10.3390/ijms160818149
- Laura Feltri, M., Suter, U., & Relvas, J. B. (2008). The function of rhogtpases in axon ensheathment and myelination. Glia, 56, 1508–1517. https://doi.org/10.1002/glia.20752
- Marinissen, M. J., Chiariello, M., Tanos, T., Bernard, O., Narumiya, S., & Gutkind, J. S. (2004). The small GTP-binding protein RhoA regulates c-jun by a ROCK-JNK signaling axis. Molecular Cell, 14, 29–41. https://doi.org/10.1016/S1097-2765(04)00153-4
- Mcbeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K., & Chen, C. S. (2004). Cell shape, cytoskeletal tenstion and RhoA regulate stem cell lineage committment. Developmental Cell, 6, 483–495. https://doi.org/10.1016/S1534-5807(04)00075-9
- Mceachern, M. J., Blackbum, E. H., Olson, M. F., Ashworth, A., & Hall, A. (1995). An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1 constitutively. Science, 269, 1270–1272.
- McMurray, R. J., Dalby, M. J., & Tsimbouri, P. M. (2015). Using biomaterials to study stem cell mechanotransduction, growth and differentiation. Journal of Tissue Engineering and Regenerative Medicine, 9(5), 528–539. https://doi.org/10.1002/term.1957
- Mouw, J. K., Ou, G., & Weaver, V. M. (2015). Extracellular matrix assembly: A multiscale deconstruction. Nature Reviews. Molecular Cell Biology, 15, 771–785. https://doi.org/10.1038/nrm3902 Extracellular
- Piccolo, S., Dupont, S., & Cordenonsi, M. (2014). The biology of YAP/TAZ: Hippo signaling and beyond. Physiological Reviews, 94, 1287–1312. https://doi.org/10.1152/physrev.00005.2014
- Poitelon, Y., Lopez-Anido, C., Catignas, K., Berti, C., Palmisano, M., Williamson, C., … Feltri, M. L. (2016). YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells. Nature Neuroscience, 19, 879–887. https://doi.org/10.1038/nn.4316
- Sailem, H., Bousgouni, V., Cooper, S., & Bakal, C. (2014). Cross-talk between Rho and Rac GTPases drives deterministic heterogeneity. Open Biology, 4, 130132. https://doi.org/10.1098/rsob.130132
- Schreck, I., Al-Rawi, M., Mingot, J. M., Scholl, C., Diefenbacher, M. E., O'Donnell, P., … Weiss, C. (2011). c-Jun localizes to the nucleus independent of its phosphorylation by and interaction with JNK and vice versa promotes nuclear accumulation of JNK. Biochemical and Biophysical Research Communications, 407, 735–740. https://doi.org/10.1016/j.bbrc.2011.03.092
- Taylor, C. A., Braza, D., Rice, J. B., & Dillingham, T. (2008). The incidence of peripheral nerve injury in extremity trauma. American Journal of Physical Medicine & Rehabilitation, 87, 381–385. https://doi.org/10.1097/PHM.0b013e31815e6370
- Totaro, A., Castellan, M., Battilana, G., Zanconato, F., Azzolin, L., Giulitti, S., … Piccolo, S. (2017). YAP/TAZ link cell mechanics to Notch signalling to control epidermal stem cell fate. Nature Communications, 8, 1–13. https://doi.org/10.1038/ncomms15206
- Turunen, S., Haaparanta, A. M., Aanismaa, R., & Kellomaki, M. (2013). Chemical and topographical patterning of hydrogels for neural cell guidance in vitro. Journal of Tissue Engineering and Regenerative Medicine, 7(4), 253–270. https://doi.org/10.1002/term.520
- Urbanski, M. M., Kingsbury, L., Moussouros, D., Kassim, I., Mehjabeen, S., Paknejad, N., & Melendez-Vasquez, C. V. (2016). Myelinating glia differentiation is regulated by extracellular matrix elasticity. Scientific Reports, 6, 1–12. https://doi.org/10.1038/srep33751
- Versaevel, M., Grevesse, T., & Gabriele, S. (2012). Spatial coordination between cell and nuclear shape within micropatterned endothelial cells. Nature Communications, 3, 671. https://doi.org/10.1038/ncomms1668
- Vertelov, G., Gutierrez, E., Lee, S. A., Ronan, E., Groisman, A., & Tkachenko, E. (2016). Rigidity of silicone substrates controls cell spreading and stem cell differentiation. Scientific Reports, 6, 1–6. https://doi.org/10.1038/srep33411
- Woodhoo, A., Alonso, M. B. D., Droggiti, A., Turmaine, M., D'Antonio, M., Parkinson, D. B., … Jessen, K. R. (2009). Notch controls embryonic Schwann cell differentiation, postnatal myelination and adult plasticity. Nature Neuroscience, 12, 839–847. https://doi.org/10.1038/nn.2323
- Xu, J., Sun, M., Tan, Y., Wang, H., Wang, H., Li, P., … Li, Y. (2017). Effect of matrix stiffness on the proliferation and differentiation of umbilical cord mesenchymal stem cells. Differentiation, 96, 30–39. https://doi.org/10.1016/j.diff.2017.07.001
