Direct and indirect manipulation of the MEK‐ERK pathway regulates the formation of a pericellular HA‐dependent matrix by chick articular surface cells without modifying CD44 expresssion
Authors: Bastow Edward R. , Wheeler‐Jones Caroline P.D. , Pitsillides Andrew A. 
Published: August 2004

Introduction  Recent evidence suggests that hyaluronan (HA) facilitates the mechano‐dependent joint cavity‐forming process through the elaboration and retention of a HA‐rich pericellular matrix in the developing joint interzone (IZ). The presumptive joint IZ phenotype shows a capacity to bind and synthesize HA and also exhibits elevated activated ERK, prior to synovial joint cavity formation (Lamb et al. 2001; Edwards et al. 1994; Dowthwaite et al. 1998). We have found that immobilization, which induces embryonic joint fusion with loss of the joint IZ phenotype, also reduces ERK activity levels in the IZ. As the signalling events regulating the synthesis and binding of HA have yet to be determined, we hypothesize that ERK activation plays a pivotal role in determining the presumptive joint IZ phenotype through HA synthetic and binding capacity.Materials and methods  Chick articular surface (AS) cells were harvested from proximal tibiotarsal joints of embryos by collagenase digestion. Pericellular coat formation was assessed using the erythrocyte exclusion assay and cell‐coat area ratios determined. ERK activity was modulated by transient transfection of GFP constructs of constitutively active (CA‐) or dominant negative (DN‐) forms of MEK, the direct upstream regulator of ERK or by treatment with the MEK inhibitor PD98059 (50 µm). ERK activation was monitored by immunochemistry. CD44 expression and ERK activation in PD98059‐treated cells were monitored by immunoblotting and medium HA concentrations by ELISA.Results  AS cells form large pericellular coats that are lost following hyaluronidase treatment and thus dependent upon HA for their construction. Treatment with PD98059 significantly reduced pericellular coat formation after 6 h. In parallel, we confirmed that PD98059 diminished active ERK expression without modifying overall levels of ERK, suggesting that the elaboration of large HA‐pericellular coats is dependent upon MEK's activation of ERK. Western blot analysis of PD98059‐treated cells showed that loss of pericellular coats was not, however, associated with any decreased levels of the cell surface HA receptor CD44. Although treatment with PD98059 did not change medium HA concentration after short times of exposure, at times (up to 6 h) during which coat loss was evident, prolonged treatment over 24 h significantly decreased medium HA concentration. Consistent with a role for ERK in pericellular coat formation, transfection with DN‐MEK diminished, while CA‐MEK increased, both active ERK expression and coat formation efficiency. We also found that, commensurate with this modification in coat forming efficiency, cells expressing DN‐MEK exhibited a significant reduction in labelling of free HA on the cell surface.Discussion  These studies extend our recent work to indicate that: (i) direct modulation of ERK activation by transfection with its endogenous upstream regulator modifies cell surface‐associated HA (ii) PD98059‐induced blockade of ERK activation restricts medium HA release and (iii) ERK‐mediated changes in pericellular coat elaboration are independent of changes in cellular CD44 expression. These findings suggest an intimate relationship between ERK activation and the formation/retention of HA‐rich pericellular matrices in vitro and highlight the role for ERK activation in regulating joint line‐related differentiation.