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 matrixproducing 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 chondrogenesispromoting 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 Juhász and Matta et al. 3 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.

PACAP and VIP signaling in chondrogenesis and osteogenesis

“…Sox9 promoter is known to be regulated by the CREB that binds to a CRE site upstream of Sox9 [30]. We have demonstrated that Sox9 and CREB transcription factors are phosphorylated by PKA during cartilage formation [31,32]. Moreover, a quite complex regulatory mechanism and synergism between Sox9 function and the cAMP-PKA-CREB pathway has been published in both mature and differentiating chondrocytes which includes BMP pathway connections [33].…”

Role of PACAP and VIP Signalling in Regulation of Chondrogenesis and Osteogenesis

“…Indeed, the addition of PACAP1-38 during oxidative stress prevented the inhibition of cartilage matrix production by normalizing the phosphorylation of Sox9 and CREB in chicken chondrogenic cells [ 13 ]. Recently, the involvement of PACAP or VIP signalling activation in mechanotransduction of developing articular cartilage has been proved by our group [ 68 ] and the importance of PKA in mechanical cellular response was proposed [ 34 ]. Mechanical load of in vitro chondrogenic cell cultures resulted in an increased PAC1 receptor and PACAP expression, and supported the undifferentiated stage of chondroblasts.…”

A novel compressive stress-based osteoarthritis-like chondrocyte system

“…A moderate level of cyclic mechanical stimulus increases the production of cartilage ECM components and promotes cartilage thickening, 25,26 whereas high levels of mechanical loading induce inflammatory responses, accelerate cartilage degradation, and eventually lead to OA. [27][28][29] In this study, expression of the genes encoding aggrecan and type I and type II collagens were upregulated at cyclic loads of 30-40 psi, but downregulated at 60 and 120 psi (Figure 2(a) and (b)).…”