It is well established that local modification of extracellular matrix (ECM) hyaluronan composition is vital in the regulation of cell behavior. Indeed, the formation of articulating chick joint cavities, which requires mechanical stimuli derived from skeletal movement, is dependent upon the accumulation of an ECM rich in hyaluronan (HA). However, the mechanisms responsible for such precise mechano-dependent regulation of cell behavior and the formation of a HA-rich ECM remain undefined. Here we show that extracellular-regulated kinase 1/2 (ERK1/2) is selectively activated in cells at sites of cavity formation and activity diminished by in ovo immobilization that induces cartilaginous fusion across presumptive joint interzones. In vitro analyses offer mechanistic support for the role of mechanical stimuli in promoting a MEK-dependent activation of ERK1/2. In addition, our direct regulation of ERK1/2 phosphorylation status via modulation of its up-stream "classical cascade" activator either pharmacologically or by transfection with dominant negative or constitutively active Mek confirms the essential role for ERK1/2 activation in the elaboration of HA-rich pericellular matrices. Together, our findings demonstrate that the MEK-ERK pathway, regulated by mechanical stimuli, controls HA-rich matrix assembly. The precision of ERK1/2 activation selectively distinguishing cells at the joint line suggests that it directly contributes to the loss of tissue cohesion essential for generating HA-rich cavities between joint elements during their development.

Pancuronium bromide (PB) is used in neonates and pregnant women to induce limp, flaccid paralysis in order to allow mechanical ventilation during intensive care. Such non-depolarizing neuromuscular blocking drugs are administered to 0.1% of all human births in the UK. In this study, we examined PB effects on skeletal development in chick embryos. PB treatment produced skeletal deformities associated with significant reduction in longitudinal growth of all appendicular elements. This was associated with greater cartilage to bone ratios, indicating a preferential reduction in osteogenesis. PB also increased the incidence of knee joint flexion and tibiotarsal joint hyperextension. In addition to limb, spinal and craniofacial deformities, flaccid immobility appears to convert the normal geometric pattern of weight gain to a simple arithmetic accretion. This novel study highlights the potentially harmful effects of pharmacologically induced flaccid immobility on chick embryonic skeletal development. Whilst in ovo avian development clearly differs from human, our findings may have implications for the fetus, premature and term neonate receiving such non-depolarizing neuromuscular blocking drugs.

Members of the fibroblast growth factor (FGF) family and growth and differentiation factor 5 (GDF-5) have been implicated in joint specification, but their roles in subsequent cavity formation are not defined. Cavity formation (cavitation) depends upon limb movement in embryonic chicks and factors involved in joint formation are often identified by their expression at the joint-line. We have sought support for the roles of FGF-2, FGF-4, and GDF-5 in cavitation by defining expression patterns, immunohistochemically, during joint formation and establishing whether these are modified by in ovo immobilisation. We found that FGF-2 exhibited low level nuclear expression in chondrocytes and fibrocartilage cells close to presumptive joints, but showed significantly higher expression levels in cells at, and directly bordering, the forming joint cavity. This high-level joint line FGF-2 expression was selectively diminished in immobilised limbs. In contrast, we show that FGF-4 does not exhibit differential joint-line expression and was unaffected by immobilisation. GDF-5 protein also failed to show joint-line selective labelling, and although immobilisation induced a cartilaginous fusion across presumptive joints, it did not affect cellular GDF-5 expression patterns. Examining changes in GDF-5 expression in response to a direct mechanical strain stimulus in primary embryonic chick articular surface (AS) cells in vitro discloses only small mechanically-induced reductions in GDF-5 expression, suggesting that GDF-5 does not exert a direct positive contribution to the mechano-dependent joint cavitation process. This notion was supported by retroviral overexpression of UDPGD, a characteristic factor involved in hyaluronan (HA) accumulation at presumptive joint lines, which was also found to produce small decreases in AS cell GDF-5 expression. These findings support a direct mechano-dependent role for FGF-2, but not FGF-4, in the cavitation process and indicate that GDF-5 is likely to influence chondrogenesis positively without contributing directly to joint cavity formation. Developmental Dynamics 235:826 -834, 2006.

Mechanisms regulating cell behavior and extracellular matrix composition in response to mechanical stimuli remain unresolved. Our previous studies have established that the MEK-ERK cascade plays a specific role in the mechano-dependent joint formation process by promoting the assembly of pericellular matrices reliant upon hyaluronan (HA) for their integrity. Here we demonstrate: (i) novel cross-talk between p38 MAPK and MEK-ERK signaling pathways that is specific for mechanical stimuli and (ii) a role for p38 MAPK in facilitating HA production by cells derived from the articular surface of embryonic chick tibiotarsal joints. We find that p38 MAPK blockade restricts pericellular assembly of HA-rich matrices and reduces basal as well as mechanical strain-induced release of HA. p38 MAPK blockers potentiated early strain-induced increases but restricted sustained increases in MEK/ERK phosphorylation at later times; c-Fos hyperphosphorylation at threonine 325 was found to parallel this p38 MAPK-mediated modulation of ERK activation. In contrast, p38 MAPK inhibitors had no detectable effect on the ERK activation induced by fibroblast growth factor 2 or pervanadate, a phosphatase inhibitor, and MEK inhibitors did not influence p38 MAPK phosphorylation, confirming both the specificity and unidirectionality of p38 MAPK-ERK cross-talk. Immunochemical and immunoblotting studies revealed constitutive p38 MAPK activation in cells at, or derived from, developing articular joint surfaces. Unlike the MEK-ERK pathway, however, p38 MAPK was not further stimulated by mechanical stimulation in vitro. Thus, p38 MAPK specifically facilitates ERK activation and downstream signaling in response to mechanical stimuli. These results suggest that constitutively active p38 MAPK serves an essential, permissive role in mechanically induced changes in ERK activation and in the accumulation of HA-rich extracellular matrices that serve a key role in joint development.Mitogen-activated protein kinases (MAPKs) 3 are ubiquitous serine/ threonine kinases that are activated by diverse extracellular stimuli, including growth factors, cytokines, and physiological mechanical signals (1-3). MAPKs are essential for transducing signals from the cell surface that regulate diverse cellular behaviors, such as those coordinating development, proliferation, and differentiation. Much recent emphasis has been placed on members of three well characterized mammalian MAPK families, comprising extracellular signal-regulated kinases (ERKs), p38 MAPKs, and c-Jun N-terminal kinases (JNKs) (4 -6). Until relatively recently it was considered that these families were preferentially activated by certain types of signal: the ERK family by growth factors and the p38 MAPK and JNK families by cellular stress. It is now becoming increasingly clear, however, that the various MAPK pathways can also be co-activated by a single stimulus. The precise interaction between members of these distinct MAPK families and how they cooperate to control cell behavior are, however, ill-define...