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Originally published In Press as doi:10.1074/jbc.M414495200 on January 12, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11749-11758, March 25, 2005
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Selective Activation of the MEK-ERK Pathway Is Regulated by Mechanical Stimuli in Forming Joints and Promotes Pericellular Matrix Formation*

Edward R. Bastow{ddagger}§, Katherine J. Lamb{ddagger}§, Jo C. Lewthwaite{ddagger}, Anne C. Osborne, Emma Kavanagh||, Caroline P. D. Wheeler-Jones{ddagger}, and Andrew A. Pitsillides{ddagger}**

From the {ddagger}Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College St., London, NW1 0TU, United Kingdom

Received for publication, December 23, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well established that an extracellular matrix (ECM)1 rich in hyaluronan (HA) modulates cell behavior such as migration, differentiation, adhesion, and proliferation and also serves a structural role by enhancing the fluidity of the ECM (1, 2). An example of such localized enrichment in HA occurs during the formation of articular joints, where separation of opposed skeletal elements requires exact spatial control (3, 4). Earliest commitment of cells to this fate is evident in the blastemal mesenchyme, between expanding cartilaginous elements, where intervening presumptive joint interzones persist in zones exhibiting restricted chondrogenesis (5–7).

Factors including Wnt-14, Gdf-5, Cux1, and stanniocalcin show specific expression in joint interzones and influence joint formation but do not account for the precision of joint cavity formation (cavitation) (8–11). The mechanisms responsible for defining the precise site of cavitation, therefore, remain unclear, and factors regulating the accumulation of HA during the formation of the cell-free joint cavity remain the subject of speculation (12, 13).

In addition to an enrichment in HA, cells of the presumptive joint also express elevated levels of CD44 (principal HA-binding protein, HABP) and moesin, an actin-capping protein, to which HA-associated CD44 binds (14, 15). Our previous finding that HA oligosaccharide-induced blockade of HA-HABP interaction blocks joint formation highlights a central role for HA in cavitation (14). Movement is crucial for joint cavitation (16–20), but how these mechanically derived signals promote the joint-forming cellular phenotype has not been addressed.

The extracellular-regulated kinases 1/2 (ERK1/2) family of mitogen-activated protein kinases can act as a point of convergence for many signals, including those induced by mechanical perturbation (21–23). These pathways convert extracellular stimuli into signals that control gene expression, cell proliferation, and differentiation. Here we examine the hypothesis that the precision of cavity formation is regulated by a mechanically induced ERK1/2 activation and that this contributes to acquisition of the joint-forming cellular phenotype. Using in vitro and in ovo approaches, we show that active ERK1/2 localizes to cells at sites of cavity formation with a precision that explains cell behavior within joint interzones. We also show that this selective activation is lost after in ovo immobilization, indicating an integral role for ERK1/2 in joint formation. Together with transfection studies showing the essential role of ERK1/2 in HA-rich pericellular matrix assembly, these findings indicate that the classical mitogen-activated protein kinase pathway is a mechanically induced determinant of cell behavior regulating the accumulation of HA during the formation of the cell-free joint cavity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Transmission and Scanning Electron Microscopy—Transmission electron microscopy was performed on stage 44 embryonic chick tibiotarsi as described previously (24, 25). For scanning electron microscopy, tissue was fixed and dehydrated as for transmission electron microscopy, dried in a critical point dryer (CPD, Samdri), mounted on aluminum stubs, coated in an Emscope SC500 sputter coater, and examined on a Hitachi S-450 scanning electron microscope.

Immobilization during Chick Limb Development—Stage 36 chick embryos were treated in ovo with decamethonium bromide for 3 days as previously described (20). Controls were treated with vehicle alone. Chicks were killed at stage 40 by decapitation (Animal Scientific Procedures act 1986). Limbs were snap-chilled in n-hexane (low in aromatic hydrocarbons, VWR International) maintained below -70 °C, and cryostat sections (10 µm) were prepared (4).

Histochemistry—Sections were stained with Alcian blue 8GX using described methods (26, 27). Immunolabeling of longitudinal control and immobilized limb sections for polymerized actin, uridine diphosphoglucose dehydrogenase (UDPGD), CD44, and moesin used, respectively, (i) 1 µg ml-1 fluorescein isothiocyanate-conjugated phalloidin (Sigma), (ii) 1:10 purified rabbit antibody raised against a bovine UDPGD C-terminal synthetic peptide (458IETIGKKVSS467; data base A54926S; a gift from Roger Mason and Mark Chambers) followed by 1:50 biotinylated goat anti-rabbit IgG in Tris-buffered saline/Tween with 20% filtered chick serum (1 h), detected using avidin-conjugated glucose oxidase complex (ABC-GO kit; Vector Laboratories) and substrate (GO-NBT substrate kit; Vector) according to the manufacturer's instructions, (iii) 1:100 mouse anti-chick CD44 (a gift from F. Davison and C. Tregaskes) visualized using glucose oxidase complex (ABC-GO kit; Vector) as described herein, and (iv) anti-hamster kidney cell moesin visualized using the appropriate streptavidin-conjugated alkaline phosphatase secondary antibody (4) and reacted in medium containing naphthol-AS-MX phosphate, levamisole, and Fast Blue BB salt. Phosphorylated ERK1/2 (New England Biolabs, catalog number 9101, rabbit polyclonal antibody against dual-phosphorylated (Thr-202/Tyr-204) ERK1/2, at 1:100 dilution) and total ERK1 (BD Transduction Laboratories, clone MK12, mouse monoclonal antibody against a peptide fragment corresponding to residues 325–345 close to the C terminus of rat ERK1, at 1:20) or total ERK2 (BD Transduction Laboratories, clone 33, mouse monoclonal raised against the entire 219–358 residue C-terminal fragment of rat ERK2, at 1:20) were detected with appropriate fluorescein isothiocyanate (1:50)- or TRITC-conjugated (1:20) antibodies in Tris-buffered saline/Tween containing 20% chick serum (28) or by alkaline phosphatase-conjugated (1:50) secondary antibodies. Controls included omission of primary antibody and replacement of primary antibody with appropriate species-matched normal sera. Alkaline phosphatase activity was disclosed using a filtered solution containing naphthol-AS-MX phosphate, levamisole, and Fast Red TR salt (4). UDPGD activity was assayed using a quantitative cytochemical method as described previously (4).

Mechanical Strain Application to Articular Surface Cells—Articular surface (AS) cells were extracted from stage 42 chick tibiotarsi by collagenase digestion (28). Primary or first passage cells seeded onto plastic culture strips (Nunc) were grown to confluency in Dulbecco's minimal essential medium containing 5% chick serum and 50 µgml-1 ascorbic acid. Cells were cultured for 18 h in Dulbecco's minimal essential medium without serum then treated with fresh medium with or without 50 µM PD98059 (Promega) for 30 min immediately before strain application. Uniaxial dynamic mechanical strain (1 Hz, 3800 microstrain) was applied for 10 min by 4-point bending of strips as previously described (29–32). This essentially imparts tensile strain in a controlled manner by bending the cell culture-treated plastic strips onto which cells have been previously seeded. Cells were incubated for the times specified, and control cells were unperturbed (static) but otherwise treated identically.

Western Blotting—ERK activation was examined in cell lysates collected 20 min and 6 and 24 h post-strain. Protein levels were measured using a BCA assay (Pierce). Proteins (40 µg) were separated by SDS-PAGE (10%) and transferred to polyvinylidene difluoride (Immobilon P), and blots were incubated with primary and horseradish peroxidase-conjugated secondary antibodies (33). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences). Membranes were stripped and reprobed with p-ERK1/2 (as above), p-MEK1/2 (New England Biolabs, catalog number 9121, rabbit polyclonal antibody against dual phosphorylated MEK, Ser-217/Ser-221), and total ERK antibodies (as above) according to manufacturer's instructions.

Microdensitometry of ERK Expression—Serial control and immobilized limb sections were labeled for p-ERK1/2, total ERK1, or ERK2 using alkaline phosphatase-conjugated secondary antibody (see above), and expression per cell was measured with a Vickers M85A scanning and integrating microdensitometer at 535 nm (4). Individual cells at cartilaginous epiphyses (zone 10 cells deep) and presumptive articular surfaces were measured; at least 15 cells at identical sites in each of at least 2 sections per joint from at least 3 chicks were evaluated. The amount of reaction product/cell was expressed as the mean integrated extinction (x100) per 30 min. Paired data from both chondrocytes and joint line cells in control and immobilized limbs were compared by Student's t test.

Erythrocyte Exclusion Assay—Erythrocyte exclusion assays, a measure of pericellular matrix area, were performed as described (34). Briefly, AS cells (1 x 105 cells/35-mm dish) were treated in serum-free Dulbecco's minimal essential medium with or without PD98059 for 6 h, fixed in 750 µl of 4% formalin containing 1 x 108 horse erythrocytes ml-1 (Sigma), and viewed with a Zeiss Axiovert 100M inverted microscope. Cell and pericellular "coat" areas were measured using a drawing arm and semiautomated image analysis (Imaging Associates KS300, V3 software), and coat:cell area ratios were calculated; ratios <1 were considered to reflect the absence of a pericellular matrix. HA-dependent matrix assembly was verified by treatment for 1 h with 6 units ml-1 Streptomyces hyalurolyticus hyaluronidase (Sigma).

Plasmids, Constructs, and Transfection—Dominant negative (DN) or constitutively active (CA) Mek1 constructs were generated by specific substitution of the serine 221 phosphorylation site to alanine and dual substitution of serine 217 and 221 to glutamic acid, respectively. These were gifts from Chris Marshall (35). These were cloned into the bicistronic GFP vector, pIRES-hr-GFP-1a (Stratagene). Transfection with pIRES-DN-Mek1, -CA-Mek1, and pIRES control vector were performed using Effectene (Qiagen, GmbH, Germany) according to the manufacturer's instructions and incubated in Dulbecco's minimal essential medium containing 5% chick serum for 24 h. After re-plating cells were incubated for 6 h with fresh medium before erythrocyte exclusion assay or p-ERK1/2 immunocytochemistry.

Immunocytochemistry—Phosphorylated ERK1/2 was detected by adaptation of previous methods (36). Briefly, cells were fixed with 10% paraformaldehyde, permeabilized in ice cold methanol, and treated with 3% bovine serum albumin in phosphate-buffered saline before exposure to 1:20 anti p-ERK1/2 (New England Biolabs) in 1% bovine serum albumin overnight at 4 °C followed by 1:1000 goat anti-rabbit Alexa 568 (Vector). p-ERK1/2 labeling intensity in cytoplasm and nuclei (propidium iodide-positive) of individual cells was assessed using a "display profile" function of a Zeiss LSM 510. Measurements were made in 10 randomly selected cells from control or strained strips with or without PD98059. Paired data from nuclear and cytoplasmic compartments of each cell were compared by the Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mechanical Loading Regulates the Phenotype of Cells at the Joint Line—Mechanical loading can modulate cell phenotype and ECM composition (20). Visualizing articular sites with differing post-hatch loading profiles shows that cells in high load-bearing sites are closely packed, with elongated dense nuclei (Fig. 1a). By comparison, cells in low load-bearing sites show rounder morphology with less dense nuclei (Fig. 1a). Evidence of intracellular polarity toward the articular surfaces was lacking, but association between load-bearing and surface cell morphology was evident.



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  		FIG. 1.
Cell-matrix organization at presumptive joints exhibits mechanical regulation. a, scanning (SEM) and transmission electron micrographs (TEM) demonstrates closer packing of cells with elongated denser nuclei and longer projections in load-bearing (L) than in nonload-bearing (NL) regions of distal tibiotarsal joint surfaces (stage 44;FC denotes fibrocartilage) (Fig. 1a). b, in ovo immobilization with decamethonium bromide between stages 36–40 modifies distribution of sulfated glycosaminoglycans (Alcian blue) and reduces joint line UDPGD (arrowhead), CD44, and moesin expression as well as UDPGD activity (arrow). Scale bar, 100 µm. c, cytochemically demonstrable UDPGD activity is lacking in cranial cartilaginous regions of Jekyll mutant zebrafish. Results are representative of observations made in all of at least 30 fused (immobilized) versus control joints.

 
We have shown that cells at cavitation sites exhibit increased UDPGD, CD44, and moesin expression (14, 4) (Fig. 1b). We, therefore, examined whether immobilization modifies such expression. We found reduced UDPGD, CD44, and moesin expression levels in immobilized limbs where cavitation was abrogated (Fig. 1b), suggesting their involvement in joint formation. Immunodetection of UDPGD may not, however, reflect enzyme activity. We, therefore, used a quantitative cytochemical assay to examine whether UDPGD activity was also modified by immobilization (4). Cells close to presumptive joint surfaces in normal limbs showed higher UDPGD activity that was lost in immobilized limbs (Fig. 1b), whereas activity in chondrocytes was unaffected. These decreases in joint line UDPGD activity are intriguing, because UDPGD is known to modulate intercellular signaling during development (37, 38). Indeed, disruption in UDPGD in zebrafish (Jekyll mutant) blocks atrioventricular differentiation by modifying the signaling boundaries involved in valve formation (39). The role of UDPGD-derived HA in boundary formation is also supported by similar cardiac valve disruption in HAS2 (HA synthase 2) knockouts, which is correctable by exogenous HA (40, 41).

In keeping with their aberrant head phenotype, Jekyll zebrafish showed reduced activity in cranial cartilage compared with controls (Fig. 1c). This confirms our UDPGD assays and shows for the first time a deficiency in the activity of this mutant Jekyll UDPGD (39). Western analysis of normal and Jekyll fish protein also confirmed UDPGD antibody specificity (data not shown). HA synthesis requires UDPGD and its downregulation commensurate with CD44, moesin, and local HA content in immobilized limbs, supports a role for increased HA synthesis and binding during cavity formation.

Joint Line-selective Cytoplasmic ERK Activation—HA binding to CD44 can regulate specific ERK signaling events (42, 43). We hypothesized that cavity formation requires mechanically dependent activation of ERK, and to identify whether ERK1/2 is a candidate for such regulation, we examined its activation in developing limbs. Selective intense labeling specific for dualphosphorylated and, therefore, active ERK (p-ERK1/2) was found only in cells at developing articular joint surfaces (Figs. 2, a–c; specificity was confirmed by immunoblotting, see Fig. 4). The absence of label in control sections from which primary antibody was omitted or replaced with appropriate speciesmatched normal sera confirmed the specificity of labeling. These findings support regulatory mechanisms involving ERK activation, HA binding, and synthesis at the joint line.



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  		FIG. 2.
ERK1/2 exhibits joint line-selective cytoplasmic activation during joint cavity formation. Immunohistochemistry for activated ERK1/2 (a, p-ERK1/2, green) in embryonic chick tibiotarsal joints selectively labels cells at developing articular surfaces, whereas propidium iodide (b, red) does not distinguish these cells from their neighbors (c, composite of a/b). Active ERK1/2 expression is predominantly cytoplasmic in cells at the developing articular surfaces but principally nuclear in neighboring chondrocytes (d–f). Joint line cells exhibit distribution consistent with a cytoskeletal association (e). Lack of p-ERK1/2 with nuclear localization at joint lines (f, arrowhead) confirms its cytoplasmic distribution, whereas p-ERK1/2 expression in chondrocytes overlapping with propidium iodide demonstrates its nuclear distribution (f, arrows). Co-localization of p-ERK1/2 (red) with polymerized actin (phalloidin, green) selectively distinguishes cells directly bordering developing joint cavities at both low (g, arrowheads) and high (h–j) magnification. Polymerized actin exhibits a broader distribution (h) than active ERK1/2 (i), and their cellular co-localization is joint line-selective in developing limbs (j, composite of h/i). Scale bar, 20 µm. Results are representative of observations made in all of at least 10 control joints.

 



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  		FIG. 4.
Mechanical strain induces MEK-dependent ERK activation in vitro. a, ERK1/2 is activated by mechanical strain and an increased ERK phosphorylation is observed with time in culture. This, in addition to strain-induced ERK1/2 activation, is diminished by treatment with the MEK inhibitor, PD98059 (50 µM). Phosphorylated MEK1/2 expression is enhanced by PD98059 treatment at times when ERK1/2 phosphorylation is most actively promoted. These results are representative of at least 20 experiments. b and c, PD98059 reduces ERK1/2 activation in vitro and diminishes mechanical strain-induced nuclear/cytoplasmic re-distribution of p-ERK1/2, revealing a MEK-dependent cytoplasmic enrichment of active ERK1/2 in response to mechanical strain. No change in total ERK (T-ERK) is evident at any time point. Results from a representative experiment (n = 2) are expressed as the mean ± S.E. relative expression in 10 cells measured from each of four control or strained strips with or without PD98059. **, p < 0.05.

 
It is known that p-ERK1/2 has both cytoplasmic and nuclear substrates (21, 22). Subcellular distribution was, therefore, examined to identify the location of potential p-ERK1/2 targets in joint line cells. P-ERK1/2 was predominantly cytoplasmic in cells at articular surfaces but principally nuclear in chondrocytes (Fig. 2, d–f). The former exhibited an elongate distribution parallel with developing surfaces, reminiscent of polymerized actin labeling (Fig. 2e); the latter is consistent with a role in phosphorylating chondrocytic transcription targets. The absence of p-ERK1/2 colocalization with propidium iodide, along with its dual labeling with polymerized actin confirmed its cytoplasmic location (Fig. 2, f and g). Indeed, polymerized actin-p-ERK1/2 colocalization was joint line-selective in developing limbs (Fig. 2, h–j). To examine whether activation of ERK is an early determinant of cavitation, we investigated temporo-spatial expression patterns and found that p-ERK1/2 is expressed before joint formation and is conserved at congruent load-bearing joint surfaces after cavitation (Fig. 3a).



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  		FIG. 3.
The cavitation-inhibitory influence of immobilization selectively diminishes ERK1/2 activation in developing joints. a, labeling for phosphorylated ERK1/2 (green) discloses strong expression in cells at sites of cavitation before (top left; propidium iodide, red) and during (top center) joint formation, and this is retained at congruent joint surfaces after cavitation (top right). ERK1, but not ERK2 (both green), exhibits increased expression at joint lines (middle center; propidium iodide, red) but is less restricted in its distribution than active ERK1/2. ERK2 exhibits diffuse labeling throughout both interzones and cartilage regions. Results are representative of observations made in all of at least 10 control joints. Scale bar, 100 µm. b, joint line-associated active ERK1/2 expression is lost in tibiotarsal joints immobilized for 3 days (stage 36–40). Results are representative of observations made in all of at least 10 immobilized and control joints. Scale bar, 100 µm. c, microdensitometric measurement of active ERK1/2, total ERK1, and ERK2 expression per cell reveals immobilization-induced decreases in p-ERK1/2 expression at joint fusion sites without changes in neighboring chondrocytes. Preferential joint line expression of ERK1 was also reduced at sites of fusion, whereas ERK2 showed neither preferential joint line expression nor modification by immobilization. Data are expressed as the mean ± S.E. for one limb in each of three chicks. At least 15 cells were measured at each site in at least two sections. ns, not significant. p < 0.05 (*) and p < 0.01 (***) compared with control (appropriate chondrocytes or unfused control joint line cells).

 
It is currently not possible to selectively discriminate between active forms of ERK1 and ERK2 using commercially available antibodies in tissue sections. We have, therefore, used antibodies raised against total ERK1 and ERK2 isoforms in an attempt to determine whether enrichment of active ERK1 or -2 at the presumptive joint line likely reflected preferential phosphorylation of one or the other ERK isoforms. The anti-ERK1 antibodies show some cross-reactivity to ERK2. In contrast, the anti-ERK2 antibodies show no reactivity toward ERK1 and are, thus, considered specific to ERK2. We found that anti-total ERK1 labeling but not that achieved using the anti-total ERK2 antibody showed significantly greater selectivity for the articular surface of the joint. The fact that anti-total ERK1 antibodies selectively label developing joint regions but may recognize both ERK1 and ERK2 isoforms coupled with the lack of any such preferential labeling with anti-total ERK2 antibodies provides evidence supporting enrichment of ERK1 in its activated form at the joint line. Thus, examination of total ERK1 and -2 expression showed that ERK1, but not ERK2, increased at developing joint surfaces, with a distribution less restricted than activated ERK1/2 (Fig. 3a).

ERK Is Activated by Mechanical Stimuli in Vivo—Immobilization blocks joint formation in the developing limb (18, 20). In addition to promoting joint fusion, we found that immobilization markedly diminished p-ERK1/2 labeling at the joint line (Fig. 3b). Examination of expression patterns alone may not, however, discriminate between local differences in cell density and modifications in the level of expression. A meaningful measure of expression/cell was, therefore, obtained by scanning and integrating microdensitometry in histologically defined "fused" and "unfused" sites. This confirmed the immobilization-induced decreases in p-ERK1/2 expression at joint fusion sites and the lack of any modification in chondrocytes (Fig. 3c). ERK1, but not ERK2, exhibits preferential joint expression in normal limbs and was also reduced by immobilization at sites of fusion (Fig. 3c). These studies suggest that mechanical influences sustain joint line-selective ERK1/2 activation.

Mechanical Stimuli Selectively Activate ERK in Vitro—We, therefore, sought mechanistic support for this by examining MEK-ERK signaling in AS cells in response to mechanical stimuli. In immunoblotting studies we found that AS cells express predominantly ERK1 (44 kDa) and that p-ERK2 and total ERK2 are virtually undetectable in AS cells; this was confirmed using anti-total ERK1 and -2 antibodies (data not shown). In our studies we found that ERK1 is rapidly activated 20 min after mechanical strain application and that this is reduced by 50 µM PD98059, a concentration that efficiently prevents activation of ERK1/2 through MEK1 and MEK2 (Ref. 44 and Fig. 4a). This concentration of PD98059 failed to significantly modify the activation status of other mitogen-activated protein kinase signaling pathways, such as p38 or c-Jun NH2-terminal kinase (not shown). In addition, strain did not phosphorylate either c-Jun NH2-terminal kinase or p38 mitogen-activated protein kinase in AS cells (not shown), indicating preferential activation of the MEK-ERK pathway in response to mechanical stimulation. Although, serum deprivation is used in other cells to restrain ERK activation (35), we find that basal MEK-dependent phosphorylation of ERK1/2 is common in serum-deprived AS cells and may mask exposure of prolonged strain-induced ERK activation (Fig. 4a). We also found that treatment with 50 µM PD98059 did not diminish p-MEK expression, but indeed, potentiated MEK activation at times when phosphorylation of ERK1/2 was most actively promoted (Fig. 4a). PD98059 prevents MEK activation by Raf and, hence, downstream ERK1/2 phosphorylation (45), yet this appears to lead to a paradoxical increase in MEK phosphorylation. This is consistent with previous studies showing that although PD98059 decreases ERK activation, it also induces Raf activation in vascular smooth muscle cells (46). We speculate that this increase in MEK phosphorylation observed in our studies is due to stimulatory feedback by poorly activated ERK on Raf or possibly due to active Raf accumulation, which cannot be de-phosphorylated before activation of its downstream effector, MEK. Thus, the enhanced MEK phosphorylation observed in the presence of this concentration of PD98059 may reflect the existence of mitogen-activated protein kinase"feedback"regulation.

To identify the location of strain-induced p-ERK1/2 within AS cells, we examined its subcellular distribution. Consistent with a cytoplasmic location of phospho-ERK1/2 in developing joint line cells and its diminution by immobilization (Fig. 3), unstimulated AS cells show greater intensity of p-ERK1/2 label in nuclei, and mechanical stimulation promotes its cytoplasmic accumulation (Fig. 4, b and c). PD98059 treatment reduced p-ERK1/2 expression in both nuclei and cytoplasm of control cells but preferentially decreased cytoplasmic p-ERK1/2 labeling in stimulated cells (Fig. 4, b and c). These results support MEK-ERK pathway involvement in the response to mechanical stimuli at the joint line.

ERK Modulates Generation of HA-rich Pericellular Matrices—Distribution of p-ERK1/2 to joint line cells in developing limbs provides a potential link between ERK1/2 activation and tissue separation, mediated by HA-HABP, during joint space generation. To determine the functional significance of ERK1/2 activation on HA-rich matrix assembly, we examined the effect of both pharmacological inhibitors of the MEK-ERK pathway and overexpressing constitutively active Mek1 (CA-Mek1) or dominant-negative Mek1 (DN-Mek1) on pericellular coat formation in vitro (34). We found that AS cells express constitutively active ERK1/2 and assemble large pericellular matrices that are hyaluronidase-sensitive (Fig. 5, a and c). Under these conditions, the MEK inhibitor PD98059 (and U0126, not shown) also diminished coat:cell ratios (Fig. 5b). This was associated with no change in total HA release into the medium at 6 h but a significant PD98059-related diminution at 24 h (Fig. 5b). This suggests that the elaboration of large HA-dependent pericellular coats depends upon MEK-ERK activation and that prolonged inhibition of MEK-ERK signaling by PD98059 also results in decreased levels of HA synthesis and release.



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  		FIG. 5.
The MEK/ERK signaling pathway is essential for hyaluronan-dependent pericellular matrix assembly. a, erythrocyte exclusion assays (EE assay) reveal that hyaluronidase (+, 6 units/ml) reduces the pericellular matrix, as indicated by reduced coat:cell ratios, compared with controls (-). b, treatment with the MEK inhibitor, PD98059 (50 µM), induces significant reduction in pericellular coat formation compared with controls (upper graph; *, p < 0.05) and changes in medium HA release at 24 but not 6 h (lower graph; *, p < 0.05). c, analyses of transfected cells using GFP bicistronic CA-Mek1 and DN-Mek1 constructs (GFP selection) demonstrates an enhancement (arrowhead) and diminution (arrow) in cellular ERK1/2 phosphorylation, respectively (control GFP-tagged pIRES constructs). Pericellular matrix formation, assessed by erythrocyte exclusion assay, is increased by CA-Mek1 transfection and reduced by DN-Mek1 transfection compared with controls (pIRES). d, transfection of AS cells in vitro with DN-Mek1 induces a commensurate reduction in coat:cell ratios, whereas CA-Mek1 produces increases in coat assembly. Coat:cell ratios were measured as described under "Experimental Procedures" in individual cells (45 per experiment) and repeated on three occasions (panels a and d). One erythrocyte is approximately equivalent to 1 µm.

 
To substantiate a role for the MEK-ERK pathway in pericellular coat formation, we examined the effects of transfection with bicistronic GFP vectors, pIRES-DN-Mek1, pIRES-CA-Mek1, and pIRES-control, which allow transfection to be monitored by GFP in single cells. Consistent with this role for the MEK-ERK pathway, transfection with DN-Mek1 diminished, whereas CA-Mek1 increased pericellular coat size (Fig. 5, c and d). Successful transfection was confirmed by appropriate modification in total MEK expression (data not shown) and in the levels of phosphorylated ERK1/2 (active-ERK) in AS cells expressing GFP; transfection with CA-Mek1 enhanced, whereas DN-Mek1 diminished active ERK1/2 levels, respectively (Fig. 5c). No change in total MEK or ERK activation status was evident in GFP-expressing control (pIRES) transfectants compared with neighboring untransfected AS cells (data not shown). These changes in pericellular coat size in response to transfection with CA-Mek1 and DN-Mek1 confirm the specificity of the influence of the MEK-ERK pathway.

To examine the dependence of coat formation upon MEK-ERK pathway activation further, we determined the dose-dependent effects of PD98059 and U0126, a structurally distinct and more potent MEK inhibitor (44), on ERK phosphorylation and pericellular coat formation. We found that both inhibitors dose-dependently reduce pericellular matrix coat size in parallel with their effects on ERK activation status (Fig. 6). Inhibitory effects were evident at 20 µM PD98059 and 1 µM U0126, consistent with their effects upon ERK phosphorylation. In contrast, the inactive isomer of U0126, namely U0124, failed to modify either ERK phosphorylation status or coat size (Fig. 6).



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  		FIG. 6.
Dose-dependent effects of MEK inhibitors on ERK activation status and pericellular coat formation. Treatment with the MEK inhibitors PD98059 (a) and U0126 (c) but not its inactive analogue U0124 (c) dose-dependently inhibit ERK activation (pERK) without modifying total ERK (tERK) expression in AS cells in vitro. Erythrocyte exclusion assays (b and d) reveal that treatment with PD98059 (b) and U0126 (d), but not U0124 (d), dose-dependently diminish pericellular matrix assembly compared with controls. Coat:cell ratios were measured (as described) in 50 cells for each treatment. Data are expressed as the mean ± S.E.

 
Together, these results suggest that signaling via the MEK-ERK pathway augments coat formation, whereas inhibition by up-stream activator blockade disrupts pericellular matrix assembly. This indicates that ERK1/2 activity status regulates HA-rich pericellular matrix formation/retention in vitro and supports a similar role for ERK1/2 in modulating cell behavior at developing joint lines. Thus, joint cavitation appears to involve site-selective mechano-dependent activation of ERK1/2, which in turn promotes elaboration of an ECM rich in cellassociated HA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our findings highlight a specific role for the MEK-ERK mitogen-activated protein kinase cascade in the joint formation process. We make the novel observations that cellular ERK1/2 activation (i) localizes precisely to sites of cavity formation, (ii) is abrogated by immobilization in ovo, (iii) is promoted in a Mek-dependent manner by mechanical stimuli in vitro, and (iv) contributes directly to the assembly of pericellular matrices that depend upon HA for their integrity. Our findings provide evidence for a direct link between cellular ERK1/2 activation via the classical mitogen-activated protein kinase pathway and elaboration of a HA-dependent pericellular matrix required for generation of cell-free joint spaces.

The importance of this is evident when joint elaboration is viewed, as it is typically, as a two-phase process; where initial patterningoflimbanlagenandinterzonesisfollowedbymechano-dependent generation of "tissue cleavage" during joint cavity formation. Factors with selective early interzonal expression are, therefore, candidate regulators of both phases (8–11). However, unequivocal identification of those cells within the interzone that generate lines of cleavage during the later phase has not, until now, been achieved. Their identification on the basis of an accumulation of active cytoplasmic ERK1/2 is likely to reflect a role for ERK1/2 in matrix adaptation by joint line cells to local tissue mechanics or a direct role in controlling HA accumulation.

Persistence of joint interzones during limb development is associated with and requires a restriction upon chondrogenesis. Our studies show that movement activates ERK1/2 in cells within these interzones at sites of joint formation. Immobilization diminishes levels of p-ERK1/2 and reduces UDPGD, CD44, and moesin expression and ultimately promotes cartilaginous interzonal joint fusion. This suggests that ERK1/2 activation is associated with the mechano-dependent induction of the joint-forming HA-synthetic phenotype and that it limits chondrogenesis. This hypothesis is supported by compelling in vitro evidence showing a MEK-dependent activation of ERK1/2 in mechanically stimulated articular surface cells and the regulation of HA-rich matrix formation by direct modulation of the MEK-ERK pathway. The recent description of the MEK-ERK-signaling pathway as a suppressor of in vitro chondrogenesis (47) is also consistent with our in vivo results, in which immobilization diminishes ERK activation and induces cartilaginous joint fusion across developing interzones. Together, this indicates that whereas mechano-dependent p-ERK1/2 inhibits chondrogenesis, it also promotes local HA production and contributes to joint cavity formation. It is, therefore, curious that active ERK1/2 co-localizes with polymerized-actin in the cytoplasm of joint line cells and that cytoplasmic accumulation is also observed in mechanically stimulated cells in vitro. This suggests that p-ERK1/2 targets cytoplasmic and/or membrane-associated proteins in these cells. However, consistent with other reports, we show that active ERK exhibits a mainly nuclear distribution in neighboring chondrocytes, where it is known to phosphorylate transcriptional targets (48, 49). It is, therefore, clear that subcellular compartmentalization of active ERK1/2 is likely to contribute directly to controlling local HA accumulation or matrix adaptation by joint line cells to local tissue mechanics.

Bobick and Kulyk (47) also showed that the MEK-ERK pathway inhibition in limb mesenchyme cells promotes a redistribution of newly synthesized collagen type II and sulfated glycosaminoglycans from pericellular to more distant matrix compartments. Our studies show that similar inhibitors of MEK-ERK signaling additionally restrict AS cell HA-dependent pericellular coat formation and suppress longer term release of HA. It is possible, therefore, to interpret these findings to suggest that the elaboration of an HA-rich pericellular matrix by chondrogenic cells is pivotal to subsequent retention of collagen type II and sulfated glycosaminoglycan within their cell-associated domains. We have previously shown, however, that AS cells derived from joint surfaces conserve a non-cartilaginous phenotype with limited sulfated glycosaminoglycan and type II collagen production in vitro (28). It is possible that their MEK-ERK-dependent elaboration of pericellular matrices, rich in HA, actively suppresses such chondrogenic differentiation. This, therefore, adds further support to the hypothesis that MEK-ERK signaling functions as an inhibitory regulator of embryonic cartilage differentiation. These considerations may also have impact on broader issues in limb development. Recent studies have led to the postulation that progenitor cells are assigned segmentally to distinct fates during early limb outgrowth (5). It is possible that the assignment of cells to chondrogenic and joint interzonal fates is, therefore, determined on the basis of such distinct cell-matrix interactions that are subject to direct regulation by MEK-ERK pathway activation status.

A link between ERK1/2 activation and formation of a HA-dependent matrix is confirmed by our Mek1 misexpression studies, showing that the MEK-ERK pathway is a vital modulator of pericellular matrix formation. This contribution may be achieved via many downstream targets, including those regulating HA synthesis, degradation, or binding. The importance of HA-receptor (CD44) binding in matrix assembly by chondrocytes and in the formation of joint cavities has been emphasized previously (14, 28, 47–49, 51). ERK1/2 may, therefore, affect coat assembly by modulating CD44 expression levels or CD44-cytoskeletal association with moesin to control HA-binding (43, 51, 52). Whether ERK activity status in AS cells is directly or indirectly responsible for modulating cell surface HA binding remains to be determined. However, it appears that although CD44 is required, it is not sufficient to confer HA binding nor are changes in its phosphorylation (53–55). The MEK-ERK pathway is, therefore, likely to modify pericellular coat formation independently of a direct CD44 phosphorylation but does not preclude changes in HA binding via CD44 dimerization, glycosylation, or cytoskeletal association (56–57). Whether mechano-dependent ERK1/2 activation modifies CD44 HA-ligand binding has not been addressed, but the changes in CD44 and moesin expression we describe herein and those showing increases in cell surface HA-binding sites in response to mechanical stimuli (12) provide support for such a mechanism.

It is common to interpret the response to immobilization and those stimulated by in vitro mechanical strain application in parallel. For example, diminution in joint line ERK activation induced by immobilization may be interpreted to directly reflect the capacity for strain-induced increases in ERK activation in vitro and lead to speculation that they are physiologically connected. This could be justified if the mechanical stimuli applied in vitro were known to correspond directly with those engendered by movement in ovo. It is currently impossible, however, to measure strains in the developing embryo. Thus, we have chosen to apply low peak strain magnitudes that are equivalent to those engendered by normal load-bearing on the surface of cortical bone, with its high resistance to bending (58). The material properties of developing joint surfaces, although undefined, are nonetheless likely to ensure that our mechanical perturbations of AS cells are within the physiological range. Despite this, it is important to stress that any such studies take little regard of the influence of cell-cell and cellmatrix interactions in the complex environment and architecture of the articular surface. Importantly, cultured AS cells exhibit a similar profile of ECM expression as they do in vivo, further justifying an examination of their response to mechanical stimuli in vitro (28).

Pericellular matrix assembly can be regulated via HA synthesis and tethering to HA synthases (HAS) at the cell surface (59–62). We have previously found that AS cells express HAS2 and that mechanical stimuli induce HAS3 mRNA and increases in UDPGD activity, as well as enhanced rates of HA secretion (28). Although matrix retention and synthesis appear to be mediated by distinct mechanisms, it remains possible that ERK activation is common to both (13, 62). Our findings indicating a reduction in levels of HA release into the medium after prolonged MEK blockade by PD98059, suggesting that inhibition of MEK-ERK signaling impacts upon both matrix assembly and HA synthesis. The specificity of these studies is further endorsed by the findings that a distinct MEK inhibitor, U0126, but not its inactive analogue also modifies matrix assembly and that the effects of both PD98059 and U0126 exhibit appropriate dose dependence corresponding to their inhibition of downstream ERK activation.

Indeed, many signals including those induced by mechanical stimuli utilize the MEK-ERK pathway (21–23, 63). Consequently, serum deprivation is often used to restrain ERK activation in vitro (35, 64). Our studies in avian AS cells and those in primary avian fibroblasts (65) indicate that unlike other cell types, such restraint is only short-lived. This is pivotal, as it has been proposed that ERK1/2 activation discriminates between signals on the basis of its strength and duration (21, 22, 66). This discrimination depends upon the selective downstream phosphorylation of the transcription factor, c-Fos (67), and this is currently being investigated.

It is also possible that kinase phosphorylation status is regulated by phosphatase activity. Mitogen-activated protein kinase (MAPK) phosphatase 3 (Mkp3)-mediated regulation of MAPK/ERK signaling in limb mesenchyme has been reported to mediate proliferative, anti-apoptotic FGF8 signaling, with the apical ectodermal ridge expressing high active ERK1/2 levels (68). This suggests that ERK1/2 serves a distinct proapoptotic function in mesenchymal cells of early limb buds. It is frequently suggested that cell death also occurs at joint lines to facilitate cavitation (69, 70), but this contention is questionable (71, 72). Indeed, recent studies fail to support a direct role for cell death in this process (73, 74). A central role for ERK1/2 in the process of joint cavitation, which occurs within blastemal mesenchyme at much later stages of limb development, indicates a likely switch to an alternative function for ERK1/2 in the elaboration of HA-rich pericellular microenvironments and a selective mechanically induced loss of tissue cohesion at the presumptive joint line. In conclusion, in contrast to the overactivation of ERK1/2 observed in pathological situations (50, 75, 76), this constitutive ERK1/2 activation that selectively distinguishes cells at the joint line is a novel example of its involvement in a precisely regulated normal developmental process that requires mechanical input and involves a local accumulation of HA-rich extracellular matrix.


   FOOTNOTES
 
* This work was supported by The Wellcome Trust, The Arthritis Research Campaign, and the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

Current address: AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, UK. Back

|| Current address: Dept. of Cell Biology, Institute of Ophthalmology, 11–43 Bath St., London, EC1V 9EL, UK. Back

** To whom correspondence should be addressed: Veterinary Basic Sciences, Royal Veterinary College, Royal College St., London, NW1 0TU, UK. Tel.: 44-20-74685036; Fax: 44-20-74685204; E-mail: apitsill{at}rvc.ac.uk.

1 The abbreviations used are: ECM, extracellular matrix; HA, hyaluronan; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular-regulated kinase; p-ERK, phosphorylated ERK; AS, articular surface; UDPGD, uridine diphosphoglucose dehydrogenase; TRITC, tetramethylrhodamine isothiocyanate; DN, dominant negative; CA, constitutively active; GFP, green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
Rabbit anti-bovine UDPGD antibody (raised against a 14 N-terminal amino acid sequence) was a kind gift from Roger Mason and Mark Chambers (Imperial College of Science and Technology, London, UK); mouse anti-chick CD44 antibody (AV6) was kindly provided by Fred Davison and Clive Tregaskes (Institute for Animal Health, Berkshire, UK); rat anti-mouse moesin antibody and Jekyll mutant (UDPGD -/-) and control zebrafish were kind gifts from Sachiko Tsukita (Kyoto University, Japan) and Didier Stanier and Emily Walsh (University of California, San Franscisco), respectively. We also thank John Bredl and Helen Hunt for technical assistance.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
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