Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures

• Published in: Cellular signalling Vol. 26; no. 3; pp. 468 - 482
• Main Authors: Juhász, Tamás, Matta, Csaba, Somogyi, Csilla, Katona, Éva, Takács, Roland, Soha, Rudolf Ferenc, Szabó, István A., Cserháti, Csaba, Sződy, Róbert, Karácsonyi, Zoltán, Bakó, Éva, Gergely, Pál, Zákány, Róza
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.03.2014
• Subjects: Aggrecans - genetics; Animals; Cartilage - growth & development; Cell Differentiation - drug effects; Cell Proliferation; Cells, Cultured; Chick Embryo; Chondrocyte differentiation; Chondrocytes - drug effects; Chondrocytes - metabolism; Chondrogenesis - drug effects; Chondrogenesis - physiology; Collagen Type II - genetics; CREB-Binding Protein - metabolism; Cyclic AMP - biosynthesis; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors; Cyclic AMP-Dependent Protein Kinases - biosynthesis; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Inhibitors - pharmacology; Epac; Extracellular matrix; Glucuronosyltransferase - genetics; Guanine Nucleotide Exchange Factors - antagonists & inhibitors; H89; Hyaluronan Synthases; Isoquinolines - pharmacology; Mechanotransduction; Mechanotransduction, Cellular - physiology; Okadaic acid; Okadaic Acid - pharmacology; Phosphorylation; Protein Kinase Inhibitors - pharmacology; Protein Phosphatase 2 - antagonists & inhibitors; Protein Phosphatase 2 - metabolism; RNA, Messenger - genetics; Signal Transduction - drug effects; SOX9 Transcription Factor - chemistry; SOX9 Transcription Factor - metabolism; Stress, Mechanical; Sulfonamides - pharmacology; PP; HDC; H89; Epac; MTT; OA; CREB; Chondrocyte differentiation; FBS; NMDA; Extracellular matrix; MSC; PBS; TRPV; PKA; PKC; PP2A; GAG; MAPK; ECM; TB; MEK; HEPES; PBST; PG; Okadaic acid; Mechanotransduction; HAS; DMMB; ERK; extracellular signal-regulated kinase; transient receptor potential receptor vanilloid; foetal bovine serum; MAPK/ERK kinase; cAMP response element binding protein; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; proteoglycan; high density cell culture; dimethylmethylene blue; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; toluidine blue; phosphoprotein phosphatase; N-methyl-d-aspartate; mitogen-activated protein kinase; protein phosphatase 2A; hyaluronan synthase; protein kinase A; glycosaminoglycan; protein kinase C; mesenchymal stem cell; exchange protein directly regulated by cAMP; phosphate buffered saline; phosphate buffered saline supplemented with 1% Tween-20
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2013.12.001

Biomechanical stimuli play important roles in the formation of articular cartilage during early foetal life, and optimal mechanical load is a crucial regulatory factor of adult chondrocyte metabolism and function. In this study, we undertook to analyse mechanotransduction pathways during in vitro chondrogenesis. Chondroprogenitor cells isolated from limb buds of 4-day-old chicken embryos were cultivated as high density cell cultures for 6days. Mechanical stimulation was carried out by a self-designed bioreactor that exerted uniaxial intermittent cyclic load transmitted by the culture medium as hydrostatic pressure and fluid shear to differentiating cells. The loading scheme (0.05Hz, 600Pa; for 30min) was applied on culturing days 2 and 3, when final commitment and differentiation of chondroprogenitor cells occurred in this model. The applied mechanical load significantly augmented cartilage matrix production and elevated mRNA expression of several cartilage matrix constituents, including collagen type II and aggrecan core protein, as well as matrix-producing hyaluronan synthases through enhanced expression, phosphorylation and nuclear signals of the main chondrogenic transcription factor Sox9. Along with increased cAMP levels, a significantly enhanced protein kinase A (PKA) activity was also detected and CREB, the archetypal downstream transcription factor of PKA signalling, exhibited elevated phosphorylation levels and stronger nuclear signals in response to mechanical stimuli. All the above effects were diminished by the PKA-inhibitor H89. Inhibition of the PKA-independent cAMP-mediators Epac1 and Epac2 with HJC0197 resulted in enhanced cartilage formation, which was additive to that of the mechanical stimulation, implying that the chondrogenesis-promoting effect of mechanical load was independent of Epac. At the same time, PP2A activity was reduced following mechanical load and treatments with the PP2A-inhibitor okadaic acid were able to mimic the effects of the intervention. Our results indicate that proper mechanical stimuli augment in vitro cartilage formation via promoting both differentiation and matrix production of chondrogenic cells, and the opposing regulation of the PKA/CREB–Sox9 and the PP2A signalling pathways is crucial in this phenomenon. [Display omitted] •Uniaxial cyclic loading enhances cartilage formation in chicken micromass cultures.•Cells respond to mechanical stimuli by substantially increased PKA activity.•Mechanical loading reduces PP2A activity.•Mechanotransduction in chondrogenesis involves cross-talk between PKA and PP2A.•Mechanical load affects phosphorylation and distribution of Sox9 and CREB.