Abstract
Inhibition of various ion channels alters chondrocyte mechanotransduction in monolayer, but the mechanisms involved in chondrocyte mechanotransduction in three- dimensional culture remain unclear. The objective of this study was to investigate the effects of inhibiting putative ion-channel influenced mechanotransduction mechanisms on the chondrocyte responses to static and dynamic compression in three-dimensional culture. Bovine articular cartilage explants were used to investigate the dose-dependent inhibition and recovery of protein and sulfated glycosaminoglycan (sGAG) syntheses by four ion-channel inhibitors: 4-Aminopyridine (4AP), a K+ channel blocker; Nifedipine (Nf), a Ca2+ channel blocker; Gadolinium (Gd), a stretch-activated channel blocker; and Thapsigargin (Tg), which releases intracellular Ca2+ stores by inhibiting ATP-dependent Ca2+ pumps. Chondrocyte-seeded agarose gels were used to examine the influence of 20 h of static and dynamic loading in the presence of each of the inhibitors. Overall, treatment with the ion-channel inhibitors had a greater effect on sGAG synthesis, with the exception of Nf, which more substantially affected protein synthesis. Treatment with Tg significantly impaired both overall protein and sGAG synthesis, with a drastic reduction in sGAG synthesis. The inhibitors differentially influenced the responses to mechanical stimuli. Dynamic compression significantly upregulated protein synthesis but did not significantly affect sGAG synthesis with Nf or Tg treatment. Dynamic compression significantly upregulated both protein and sGAG synthesis rates with Gd treatment. There was no significant stimulation of either protein or sGAG synthesis by dynamic compression with 4AP treatment. Interruption of many ion-channel signaling mechanisms affected sGAG synthesis, suggesting a complicated, multi-pathway signaling process. Also, Ca2+ signaling may be critical for the transduction of mechanical stimulus in regulating sGAG synthesis. This modulation potentially occurs through direct interactions with the extracellular matrix.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bird JL, Platt D, Wells T, May SA, Bayliss MT (2000) Exercise-induced changes in proteoglycan metabolism of equine articular cartilage. Equine Vet J 32:161–163
Burton-Wurster N, Vernier-Singer M, Farquhar T, Lust G (1993) Effect of compressive loading and unloading on the synthesis of total protein, proteoglycan, and fibronectin by canine cartilage explants. J Orthop Res 11:717–729
Buschmann MD, Gluzband YA, Grodzinsky AJ, Hunziker EB (1995) Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. J Cell Sci 108(Pt 4):1497–1508
Carter DR, Beaupre GS, Wong M, Smith RL, Andriacchi TP, Schurman DJ (2004) The mechanobiology of articular cartilage development and degeneration. Clin Orthop Relat Res S69–S77
Clark CC, Iannotti JP, Misra S, Richards CF (1994) Effects of thapsigargin, an intracellular calcium-mobilizing agent, on synthesis and secretion of cartilage collagen and proteoglycan. J Orthop Res 12:601– 611
Davisson T, Kunig S, Chen A, Sah R, Ratcliffe A (2002) Static and dynamic compression modulate matrix metabolism in tissue engineered cartilage. J Orthop Res 20:842–848
DiBattista JA, Dore S, Morin N, Abribat T (1996) Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: role of calcium and protein kinase A and C. J Cell Biochem 63:320–333
Duncan RL, Hruska KA, (1994) Chronic, intermittent loading alters mechanosensitive channel characteristics in osteoblast-like cells. Am J Physiol 267:F909-F916
Enobakhare BO, Bader DL, Lee DA (1996) Quantification of sulfated glycosaminoglycans in chondrocyte/alginate cultures, by use of 1,9-dimethylmethylene blue. Anal Biochem 243:189–191
Farndale RW, Sayers CA, Barrett AJ (1982) A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect. Tissue Res 9:247–248
Greisberg JK, Wolf JM, Wyman J, Zou L, Terek RM (2001) Gadolinium inhibits thymidine incorporation and induces apoptosis in chondrocytes. J Orthop Res 19:797–801
Guilak F, Zell RA, Erickson GR, Grande DA, Rubin CT, McLeod KJ, Donahue HJ (1999) Mechanically induced calcium waves in chondrocytes are inhibited by gadolinium and amiloride. J Orthop Res 17:421–429
Haapala J, Lammi MJ, Inkinen R, Parkkinen JJ, Agren UM, Arokoski J, Kiviranta I, Helminen HJ, Tammi MI (1996) Coordinated regulation of hyaluronan and aggrecan content in the articular cartilage of immobilized and exercised dogs. J Rheumatol 23:1586–1593
Lai CC, Hong K, Kinnell M, Chalfie M, Driscoll M (1996) Sequence and transmembrane topology of MEC-4, an ion channel subunit required for mechanotransduction in Caenorhabditis elegans. J Cell Biol 133:1071–1081
Lipman JM (1989) Fluorophotometric quantitation of DNA in articular cartilage utilizing Hoechst 33258. Anal Biochem 176:128–131
Mauck RL, Soltz MA, Wang CC, Wong DD, Chao PH, Valhmu WB, Hung CT, Ateshian GA (2000) Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng 122:252–260
Mow VC, Kuei SC, Lai WM, Armstrong CG (1980) Biphasic creep and stress relaxation of articular cartilage in compression? Theory and experiments. J Biomech Eng 102:73–84
Perkins GL, Derfoul A, AstA, Hall DJ (2005) An inhibitor of the stretch-activated cation receptor exerts a potent effect on chondrocyte phenotype. Differentiation 73:199–211
Ragan PM, Badger AM, Cook M, Chin VI, Gowen M, Grodzinsky AJ, Lark MW (1999) Down-regulation of chondrocyte aggrecan and type-II collagen gene expression with increases in static compression magnitude and duration. J Orthop Res 17:836–842
Smith CL, MacDonald MH, Tesch AM, Willits NH (2000) In vitro evaluation of the effect of dimethyl sulfoxide on equine articular cartilage matrix metabolism. Vet Surg 29:347–357
Tanaka N, Ohno S, Honda K, Tanimoto K, Doi T, Ohno-Nakahara M, Tafolla E, Kapila S, Tanne K (2005) Cyclic mechanical strain regulates the PTHrP expression in cultured chondrocytes via activation of the Ca2+ channel. J Dent Res 84:64–68
Tavernarakis N, Driscoll M (1997) Molecular modeling of mechanotransduction in the nematode Caenorhabditis elegans. Annu Rev Physiol 59:659–689
Tavernarakis N Driscoll M (2001) Mechanotransduction in Caenorhabditis elegans: the role of DEG/ENaC ion channels. Cell Biochem Biophys 35:1–18
Valhmu WB, Raia FJ (2002) myo-Inositol 1,4,5-trisphosphate and Ca(2+)/calmodulin-dependent factors mediate transduction of compression-induced signals in bovine articular chondrocytes. Biochem J 361:689–696
Wohlrab D, Lebek S, Kruger T, Reichel H (2002) Influence of ion channels on the proliferation of human chondrocytes. Biorheology 39:55–61
Wohlrab D, Wohlrab J, Reichel H, Hein W (2001) Is the proliferation of human chondrocytes regulated by ionic channels? J Orthop Sci 6:155–159
Wu LN, Ishikawa Y, Sauer GR, Genge BR, Mwale F, Mishima H, Wuthier RE (1995) Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization. J Cell Biochem 57:218–237
Wu QQ, Chen Q (2000) Mechanoregulation of chondrocyte proliferation, maturation, and hypertrophy: ion-channel dependent of matrix deformation signals. Exp Cell Res 256:383–391
Yellowley CE, Jacobs CR, Donahue HJ (1999) Mechanisms to fluid-flow-induced Ca2+ mobilization in articular chondrocytes. J Cell Physiol 180:402–408
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mouw, J.K., Imler, S.M. & Levenston, M.E. Ion-channel Regulation of Chondrocyte Matrix Synthesis in 3D Culture Under Static and Dynamic Compression. Biomech Model Mechanobiol 6, 33–41 (2007). https://doi.org/10.1007/s10237-006-0034-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10237-006-0034-1