title: Stepwise mechanical stretching inhibits chondrogenesis through cell-matrix adhesion mediated by integrins in embryonic rat limb-bud mesenchymal cells.
abstract: Biomechanical forces are major epigenetic factors that determine the form and differentiation of skeletal tissues, and may be transduced through cell adhesion to the intracellular biochemical signaling pathway. To test the hypothesis that stepwise stretching is translated to molecular signals during early chondrogenesis, we developed a culture system to study the proliferation and differentiation of chondrocytes. Rat embryonic day-12 limb buds were microdissected and dissociated into cells, which were then micromass cultured on a silicone membrane and maintained for up to 7 days. Stepwise-increased stretching was applied to the silicone membrane, which exerted shearing stress on the cultures on day 4 after the initiation of chondrogenesis. Under stretched conditions, type II collagen expression was significantly inhibited by 44% on day 1 and by 67% on day 2, and this difference in type II collagen reached 80% after 3 days of culture. Accumulation of type II collagen protein and the size of the chondrogenic nodules had decreased by 50% on day 3. On the other hand, expression of the non-chondrogenic marker fibronectin was significantly upregulated by 1.8-fold on day 3, while the up-regulation of type I collagen was minimal, even by day 3. The downregulation in the expression of chondrogenic markers was completely recovered when cell-extracellular matrix attachment was inhibited by Gly-Arg-Gly-Asp-Ser-Pro-Lys peptide or by the application of blocking antibodies for alpha2, alpha5 or beta1 integrins. We conclude that shearing stress generated by stepwise stretching inhibits chondrogenesis through integrins, and propose that signal transduction from biomechanical stimuli may be mediated by cell-extracellular matrix adhesion.
authors.affiliation: Tohoku University Graduate School of Dentistry, Aoba-ku 980-8575, Sendai, Japan derak@mail.tains.tohoku.ac.jp
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Onodera
authors.forename: Ichiro
authors.initials: I
authors.lastname: Takahashi
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Sasano
authors.forename: Jin-Wan
authors.initials: JW
authors.lastname: Bae
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Mitani
authors.forename: Manabu
authors.initials: M
authors.lastname: Kagayama
authors.forename: Hideo
authors.initials: H
authors.lastname: Mitani
mesh_terms: D000423:Alcian Blue / Q000494:pharmacology; D000818:Animals; D001696:Biomechanical Phenomena; D015153:Blotting, Western; D002448:Cell Adhesion; D049109:Cell Proliferation; D021982:Cell-Matrix Junctions; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D020219:Chondrogenesis; D003094:Collagen / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D046508:Culture Techniques; D005353:Fibronectins / Q000378:metabolism; D016023:Integrins / Q000378:metabolism; D018878:Limb Buds / Q000196:embryology*; D008648:Mesoderm / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D012825:Silicon / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D024042:Collagen Type I; D024043:Collagen Type II; D005353:Fibronectins; D016023:Integrins; D009842:Oligopeptides; D010455:Peptides; C086456:glycyl- arginyl-glycyl-aspartyl-seryl-prolyl-lysine; D003094:Collagen; D000423:Alcian Blue; D012825:Silicon