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

Abstract

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 6 days. 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 (0.05 Hz, 600 Pa; for 30 min) 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.

Keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CREB; Chondrocyte differentiation; DMMB; ECM; ERK; Epac; Extracellular matrix; FBS; GAG; H89; HAS; HDC; HEPES; MAPK; MAPK/ERK kinase; MEK; MSC; MTT; Mechanotransduction; N-methyl-d-aspartate; NMDA; OA; Okadaic acid; PBS; PBST; PG; PKA; PKC; PP; PP2A; TB; TRPV; cAMP response element binding protein; dimethylmethylene blue; exchange protein directly regulated by cAMP; extracellular matrix; extracellular signal-regulated kinase; foetal bovine serum; glycosaminoglycan; high density cell culture; hyaluronan synthase; mesenchymal stem cell; mitogen-activated protein kinase; okadaic acid; phosphate buffered saline; phosphate buffered saline supplemented with 1% Tween-20; phosphoprotein phosphatase; protein kinase A; protein kinase C; protein phosphatase 2A; proteoglycan; toluidine blue; transient receptor potential receptor vanilloid.