Regulation of chondrocyte differentiation by actin-severing protein adseverin

Abstract

Importance of actin organization in control of chondrocyte phenotype is well established, but little is known about the cytoskeletal components regulating chondrocyte differentiation. Previously we have observed up-regulation of an actin-binding gelsolin-like protein in hypertrophic chondrocytes. We have now identified it as adseverin (scinderin). Adseverin is drastically up-regulated during chondrocyte maturation, as shown by Northern blot analysis, in situ hybridization, and real-time RT-PCR. Its expression is positively regulated by the PKC and MEK signaling as shown by inhibitory analyses. Over-expression of adseverin in non-hypertrophic chondrocytes causes rearrangement of actin cytoskeleton, a change in cell morphology, a dramatic (3.5-fold) increase in cell volume, and up-regulation of Indian hedgehog (Ihh) and of collagen type X - all indicative of chondrocyte differentiation. These changes are mediated by ERK1/2 and p38 kinase pathways. Thus, adseverin-induced rearrangements of actin cytoskeleton may mediate the PKC-dependent activation of p38 and Erk1/2 signaling pathways necessary for chondrocyte hypertrophy, evidenced by changes in cell morphology, increase in cell size, and expression of the chondrocyte maturation markers. These results demonstrate that interdependence of cytoskeletal organization and chondrogenic gene expression is regulated at least in part by actin-binding proteins, such as adseverin

INTRODUCTION

Endochondral bone formation is a multi-step process during which condensed mesenchymal cells differentiate into hypertrophic, non-proliferating chondrocytes. The extracellular matrix surrounding the hypertrophic chondrocytes becomes calcified and replaced with bone ((Stocum et al., 1979); (Poole et al., 1989)). Chondrocyte differentiation is a precisely regulated process that involves proliferation, hypertrophic differentiation, mineralization, and apoptosis. Disruption of these events can result in various skeletal diseases and growth disorders (Zelzer and Olsen, 2003). The control of chondrocyte differentiation involves multiple extracellular and intracellular signaling molecules (reviewed in (Karsenty and Wagner, 2002); (You et al, 2001); (Haroon et al, 1999); (Nakajima et al, 2004)).)

In vivo, chondrocyte differentiation is accompanied by both drastic changes in cell shape - from a fibroblastic to a round morphology (von der Mark and von der Mark, 1977)- and a significant increase in cell volume (Wilsman et al., 1996). The increase in cell diameter is one of the first indicators of chondrocyte maturation and in vivo it precedes terminal differentiation by several days (Hirsch et al, 1996). A change in cell shape (rounding up), usually associated with a decreased cell-substratum interaction, is required for induction and/or maintenance of the chondrocyte phenotype, as has been demonstrated in numerous in vitro studies (reviewed in (Daniels and Solursh, 1991) and in (Cancedda et al., 1995); (Benya and Shaffer, 1982); (Glowacki et al., 1983)). Chondrocyte phenotype is affected by cell shape indirectly, and in fact is regulated by actin cytoskeleton ((Zanetti and Solursh, 1984); (Brown and Benya, 1988)). Further studies have confirmed the importance of actin organization in control of chondrocyte phenotype. For example, actin-disrupting compounds, such as cytochalasin D, stimulate chondrogenesis ((Loty et al, 1995); (Woods, et al, 2005)). However, to date, the molecular mechanisms responsible for the interdependence of chondrocyte differentiation status and actin organization are largely unknown, and surprisingly little is known about the chondrocyte-specific intracellular regulators of cytoskeletal rearrangements.

In various cell types the dynamics of filamentous actin (F-actin) is orchestrated by a variety (more than 60 classes) of actin-binding proteins that function downstream of the Rho-family GTPases (reviewed in (Tojima and Ito, 2004)). Many of these proteins are ubiquitously expressed, while others are up-regulated at a particular developmental stage or in a tissue-specific manner. Previously, we reported an up-regulation of a gelsolin-like protein in hypertrophic versus non-hypertrophic chondrocytes (Nurminskaya and Linsenmayer, 1996). Gelsolin superfamily of actin-binding proteins includes gelsolin, villin, adseverin (also named scinderin), capG, advillin, supervillin and flightless I. Members of this family control actin organization by severing filaments, capping filament ends and nucleating actin assembly in a calcium-dependent manner (Yin, 1987). They are found in a wide range of vertebrate, lower eukaryotic and plant cells, and their functions vary significantly: from remodeling actin filaments to specific non-overlapping roles in various cellular processes controlling cell motility, apoptosis, phagocytosis and even gene regulation (reviewed in (Kwiatkowski, 1999); (Silacci et al., 2004); (McGough et al., 2003)).

In the present study we show that adseverin (scinderin) is dramatically up-regulated during chondrocyte maturation, and in vivo expression of adseverin is restricted to the prehypertrophic and hypertophic cartilage of the embryonic growth plate. On the contrary, gelsolin expression remains constant during chondrocyte differentiation. We further demonstrate that expression of adseverin is controlled by the Ca2+-dependent protein kinase pathway (PKC), known to be essential to maintain chondrogenic phenotype (Yoon et al., 2002). Elevated levels of adseverin result in a significant (over 3-fold) increase in chondrocyte cell size. Moreover, adseverin acts as an important regulator of chondrocyte differentiation, and over-expression of this protein in non-hypertrophic chondrocytes is sufficient to cause a decrease in cell proliferation and to promote differentiation, as evidenced by up-regulation of Indian hedgehog (Ihh) and collagen type X. These changes are accompanied by activation of the mitogen-activated protein (MAP) kinases ERK1/2 and p38, the known regulators of chondrogenesis ((Zhen et al., 2001); (Bobick and Kulyk, 2004)). Our data suggest that in chondrocytes, adseverin regulates the onset of hypertrophy in a MAPK-dependent manner. The proposed role of adseverin in the regulation of cell differentiation is not limited to chondrocytes: recently it has been implicated in differentiation of megakaryoblastic leukemia cells (Zunino et al., 2001). Thus, the actin-binding proteins, and adseverin in particular, represent an integral part of the differentiation programme in a number of cell lineages.

MATERIALS AND METHODS

Chondrocyte Culture

Cartilages were dissected from 14-d-old chicken embryos (Spafas Inc., Norwich, CT). Hypertrophic chondrocytes were from the hypertrophic zone three (Schmid and Conrad, 1982) of the tibia. Non-hypertrophic chondrocytes were from the caudal one-third of the sternum. We employ sternum in our studies as the tissue source of non-hypertrophic chondrocytes, because non-hypertrophic cells from the resting zone of the growth plate differentiate and initiate collagen type X very rapidly in cultures. A significant number of collagen type X -positive cells (up to 20%) can be detected immunohistologically in the overnight cultures of resting chondrocytes (Nurminskaya et al., 2003). Cells were maintained as previously described (Nurminskaya et al., 1998). To initiate differentiation of non-hypertrophic chondrocytes ascorbic acid (50 µg/ml) was added to the culture medium ((Tacchetti et al., 1987); (Gerstenfeld and Landis, 1991)), which was changed every other day over the period of five days. 3-D collagen gels were made from Vitrogen-100 solution (Cohesion, CA) according to manufactures’ instructions.

Kinase inhibitors were obtained from Calbiochem (San Diego, CA). All drugs were dissolved in dimethyl sulfoxide (DMSO) and preserved. The final concentration of DMSO was ≤0.1%. The final concentrations of kinase inhibitors were as following: p38 inhibitor SB202190 at 15 µM, MEK inhibitor U0126 at 5 µM, PKC inhibitor bisindolylmaleilide at 1µM.

Preparation of RNA, Northern Analysis and real-time PCR

Total RNA was isolated with Trizol reagent (GIBCO BRL). RNA samples (20 µg) were subjected to electrophoresis through 1% agarose/2.2 M formaldehyde gels and transferred to a HybondN filter (Amersham Corp.). Hybridization was performed as described previously (Nurminskaya et al., 1998). First-strand cDNA was synthesized with SuperScript III (Invitrogen) using 0.5–1 µg of total RNA and oligo-(dT) primer. 1/50 part of the 15 µl reverse transcription reaction was used as template for 25 µl Real-time PCR reaction. PCR reactions were performed in triplicates in the 5700 Sequence Detector (ABI) using the QuantiTect SYBR Green PCR mix (Qiagen).

In Situ Hybridization

Tibiotarsi from 14- day -old chicken embryos (Spafas Inc., Norwich, CT) fixed for 3 h in 4% PFA were embedded in paraffin wax. Tissues were cut into 6-µm sections and mounted on Vectabond-coated slides (Vector Laboratories, Burlingame, CA). Preparation of riboprobes and hybridization were performed as previously described (Nurminskaya et al., 1998). Signal was detected with alkaline phosphatase conjugated anti-digoxigenin antibody (Boehringer Mannheim Corp., Indianapolis, IN) and NBT/BCIP.

5'- and 3'-RACE, DNA Sequencing

To extend the lengths of the partial cDNA sequence for gelsolin-like protein, isolated by subtractive hybridization (Nurminskaya and Linsenmayer, 1996), 5'-and 3'-RACE was used, using the Marathon cDNA Amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The extended, amplified products were cloned into the plasmid pCRII (TA cloning kit; Invitrogen). For each RACE product, 24–48 colonies were picked and amplified by PCR using the same insert-specific primers. The positive clones were then analyzed for insert size by PCR amplification with vector primers. The longest clones for 5'-and 3'-RACE products were sequenced from both strands after obtaining sets of gamma-delta transposon insertions (Strathmann et al., 1991). Sequencing was performed in the 373A automated DNA sequencer (PE Applied Biosystems, Foster City, CA) using the DyePrimer Cycle Sequencing kits. The results were analyzed with the Sequencher software (GeneCodes Corp., Ann Arbor, MI). The 3.12-kb full-length cDNA sequence was submitted to GenBank accession no. DQ020143.

Vector Construction, Transfection, and Immunochemical Analyses

An adseverin-red fluorescent protein expression vector (pAdseverin-Red) was constructed using the pDsRed vector (Clontech). Transient transfections were performed using Fugene reagent (Roche). 7 × 10⁴ hypertrophic chondrocytes or 7 × 10³ non-hypertrophic chondrocytes were transfected with 1 µg of DNA according to the manufacturer's instructions. At intervals after transfection, the transfected cultures were analyzed by immunofluorescence and immunoblotting. For immunofluorescence, the cells were fixed in 4% PFA for 25 min at 4°C, permeabilized by methanol, and then reacted with an anti-type X collagen mAb X-AC9 (Schmid and Linsenmayer, 1985), followed by FITC-conjugated secondary anti-mouse antibodies. For analysis of filamentous actin cells were fixed with ice-cold acetone and stained with FITC-labeled phalloidin (Sigma). The nuclei were visualized by Hoechst staining.

Immunoblotting

For immunoblotting, the cell cultures of non-hypertrophic chondrocytes transfected were either pAdseverin-Red or the control original pDsRed were sorted by FACS. Equal aliquots of transfected cells were used for analysis of monomeric/fibrillar actin, as described (Beaulieu et al., 2005). Briefly, monomeric actin was extracted with PBS by the freeze/thaw cycle. The remaining filamentous actin was extracted with 1% Triton X-100. Protein samples were analyzed for actin by Western blot. The monoclonal antibody JLA20 developed by Dr. Lin was obtained form the Developmental Studies Hybridoma Bank (developed under the auspices of the NICHD and maintained by The University of Iowa).

Analysis of MAP kinase pathway in the sorted transfected non-hypertrophic chondrocytes was performed with p44/42 MAP kinase, SAPK/JNK, p38 MAP kinase, phospho-p44/42 MAP kinase (Thr202/204), phospho-SAPK/JNK (Thr183/185), phosphor-p38 (Thr180/182) antibodies obtained from Cell Signaling (Beverly, MA). Beta-tubulin was detected with monoclonal TUB 2.1 antibody (Sigma).

RESULTS

1. Cloning of chicken adseverin

In our previous survey of genes differentially expressed in hypertrophic versus non-hypertrophic chondrocytes (Nurminskaya and Linsenmayer, 1996), we described a 160 bp cDNA fragment coding for the gelsolin family protein. To further characterize this protein expressed in hypertrophic chondrocytes, we obtained the full-length cDNA from this cell type by the RACE (Rapid Amplification of cDNA Ends) with the gene-specific primers designed using the original 160 bp sequence. The resulting overlapping clones comprised a 3.12 kb-full-length cDNA (GenBank accession no. DQ020143). The 2169 bp open reading frame encodes a protein of 722 amino acid residues, which displays the six repeated, homologous conserved domains (G1–G6) characteristic for the gelsolin family proteins ((Kwiatkowski et al., 1986); (Way and Weeds, 1988)). Because the protein shares higher levels of homology with mammalian adseverins (overall homology of 78% with mouse adseverin and 81% with bovine adseverin) than with the chicken gelsolin (61%), we identify the cloned molecule as chicken adseverin.

Earlier studies have shown that in mammalian adseverin (also named scinderin) the G1 domain contains the G-actin-binding sites while the G2 domain is responsible for the actin filament binding essential for the actin-severing activity (Sakurai et al., 1991). Chicken adseverin most likely possesses the same actin-binding properties as mammalian adseverin, based on the 89% homology between the G1 and G2 domains of the avian and the mouse proteins (Fig. 1A). However, cell type-specific forms of adseverin with altered ability to regulate actin cytoskeleton have been described, for example, in lymphocytes (Robbens et al., 1998). To evaluate the potential of chicken adseverin expressed in chondrocytes to affect the actin cytoskeleton, we over-expressed a fusion protein comprising the adseverin and the fluorescent DsRed in chondrocytes by transient transfections, and analyzed F-actin in the transfected cells using phalloidin, a probe for filamentous actin (Fig. 1B). Elevated levels of adseverin caused a significant reduction in F-actin as compared to non-transfected cells, suggesting that chicken adseverin is able to sever actin filaments, similar to its mammalian homolog.

Figure 1. Actin filament-severing activity of chicken adseverin.

A – The adseverin’s repeating domains (G1–G6) are shown in comparison with the equivalent domains of mouse adseverin. The lower numbers indicate the homology between two proteins (in %). Positions of gelsolin-like domains are numbered along the protein molecule. B - Over-expression of adseverin results in rearrangement of actin cytoskeleton as evidenced by significant reduction of filamentous actin in hypertrophic chondrocytes over-expressing Adseverin-Red fusion protein. Adseverin-DsRed was detected by red fluorescence while filamentous actin was detected with FITC-labeled phalloidin. C – Over-expression of adseverin in non-hypertrophic chondrocytes increases levels of actin monomers. Monomeric and fibrillar actin was isolated by sequential extraction from the cells expressing either DsRed protein or Adseverin-Red fusion proteins. Lysates of equal cell number analyzed by Western blot.

Further, we analyzed the ratio of monomeric to fibrillar actin in non-hypertrophic chondrocytes over-expressing adseverin by Western-blot analysis. Cells were transiently transfected with the expression vector encoding either Adseverin/DsRed fusion protein (pAdseverin-DsRed), or DsRed protein alone as a control. Transfected cells were selectively amplified by 7 days of growth in the G418-supplemented medium, and then collected by fluorescent cytometry using the DsRed fluorescent marker. Aliquots containing equal numbers of cells were allowed to attach to cell culture plates, and the monomeric and the filamentous actin forms were subsequently extracted (Beaulieu et al., 2005). Western-blot analysis confirmed a 55 (+/−6) % increase in monomeric actin in cells over-expressing the Adseverin-DsRed fusion protein versus cells over-expressing the control DsRed protein, consistent with the observed adseverin-induced disruption of actin filaments detected by fluorescence microscopy(Fig. 1C).

2. Up-regulation of adseverin during the chondrocyte hypertrophy requires PKC signaling

Initially, adseverin was identified by subtractive hybridization as one of the genes up-regulated in avian hypertrophic versus non-hypertrophic chondrocytes (Nurminskaya and Linsenmayer, 1996). In these studies, we have used the lower 1/3 of sterna as a source for the non-hypertrophic chondrocytes, while the hypertrophic chondrocytes originated from the hypertrophic zone of the tibial growth plate. To further confirm an increase in adseverin expression during chondrocyte maturation to hypertrophy, we employed several methods. The Northern blot analysis of mRNA from the cultured non-hypertrophic sternal (NH) and from the hypertrophic (Hyp) cells detected a 3.2 kb adseverin transcript only in the hypertrophic chondrocytes (Fig. 2A). On the contrary, gelsolin (another member of the gelsolin protein family) is expressed at the same levels in both cell types. Real-time PCR analysis of cells freshly isolated from the cartilaginous tissues demonstrated a dramatic 340(+/−37) - fold increase of adseverin mRNA in the hypertrophic chondrocytes as compared to the non-hypertrophic cells (normalized to beta-actin mRNA).

Figure 2. Expression of adseverin is restricted to hypertrophic chondrocytes of the embryonic growth plate.

A – Northern blot analysis of mRNA from cultured non-hypertrophic (NH) and hypertrophic chondrocytes (Hyp).The constitutive gene GAPDH was used as a loading control. B – in situ hybridization of the 14 days old embryonic growth plate with antisense adseverin RNA probe.

To substantiate that these data, obtained on the cultured cells, also correspond to the expression pattern of adseverin in vivo, we performed in situ hybridization on the growth plate of the 14 days old chicken embryos. A much stronger signal for adseverin mRNA was detected in the pre-hypertrophic and hypertrophic zones versus the resting and proliferative zones of the embryonic growth plate (Fig. 2B). Thus, adseverin expression is very low (if detectable at all) in the non-hypertrophic cartilage from two tissue sources – sternum and tibia. Taken together, these data demonstrate that adseverin is specifically up-regulated in the pre-hypertrophic and hypertrophic chondrocytes during the embryonic development of long bones.

The protein kinase C (PKC) signaling is essential for maintenance of the chondrogenic phenotype (Yoon et al., 2002). To determine whether adseverin expression during chondrocyte hypertrophy is regulated by the PKC, we analyzed the expression of adseverin in differentiating chondrocytes in which PKC activity was blocked with highly selective PKC inhibitor bisindolylmaleimide I. Non-hypertrophic chondrocytes were stimulated to differentiate in the presence of ascorbic acid ((Tacchetti et al., 1987); (Gerstenfeld and Landis, 1991)), and treated with bisindolylmaleimide I. Expression of adseverin and of other characteristic markers of chondrocyte differentiation was analyzed by real-time PCR. Two chondrogenic markers were studied: Indian hedgehog (Ihh), characteristic for pre-hypertrophic chondrocytes (Wallis, 1996), and collagen type X, specific for hypertrophic chondrocytes (Schmid and Linsenmayer, 1985). Chondrocyte differentiation was assessed morphologically by acquisition of polygonal cell shape (Fig. 3A) and at the molecular level by up-regulation of Ihh, collagen type X, and adseverin (Fig. 3B, hatched bars). Inhibition of PKC with bisindolylmaleimide I caused dedifferentiation of chondrocytes, as evidenced by fibroblastic cell morphology (Fig. 3A) and total inhibition of Ihh and collagen type X expression (Fig. 3B, black bars), in agreement with the earlier studies (Oh et al., 2000). Similarly, expression of adseverin was averted in bisindolylmaleimide-treated chondrocytes. Thus, adseverin up-regulation is specifically linked to the chondrocyte maturation program, and is regulated by the PKC similar to other characteristic markers of the pre-hypertrophic and hypertrophic chondrocytes.

Figure 3. Adseverin expression is regulated by PKC activity.

A – ascorbic acid-induced differentiation of chondrocytes is prevented by PKC inhibitor bisindolylmaleimide I, as evidenced by cell morphology. B – changes in expression of Ihh, adseverin and collagen type X evaluated by real-time PCR and normalized to beta-actin mRNA. Gene expression in non-hypertrophic chondrocytes stimulated to differentiate with ascorbic acid (hatched bars) and in ascorbic acid-treated cells grown in the presence of PKC inhibitor bisindolylmaleimide I (black bars) were compared to mRNA levels in non-stimulated non-hypertrophic chondrocytes (white bars).

(NH) and hypertorphic cells (Hyp),

3. Overexpression of adseverin promotes changes in chondrocyte morphology characteristic for hypertrophy

During hypertrophy, chondrocytes of the growth plate undergo a 10–15-fold increase in volume, and this increase in cell size can account for as much as 80% of the total increase in bone length over 24 h ((Wilsman et al., 1996); (Farnum et al., 2002)). These changes in cell volume are likely to be associated with (or probably, caused by) rearrangement of the cytoskeleton. Since adseverin is the first actin-binding protein identified as specifically up-regulated in hypertrophic chondrocytes, we analyzed the ability of this protein to cause a cell size increase. In parallel, we assessed the effect of actin-disrupting chemical agents latrunculin and cytochalasin D. Chondrocytes were transfected with a pAdseverin-DsRed or the control pDsRed expression vectors and grown in 3D-collagen gels for 7 days. The 3D gels were employed for the analyses of cell volume because in previous studies no visible morphological changes were detected in cells cultured on glass slides upon cytochalasin D-induced disruption of F-actin (Pritchard and Guilak, 2004). In addition, blockade of actin polymerization exhibits context-specific regulation of chondrogenic gene expression and results in the difference of effects in 3D- and monolayered cultures (Woods and Beier, 2006). The diameter of transfected cells was analyzed by fluorescent microscopy. Over-expression of adseverin resulted in a significant ca. 50% increase in cell diameter, corresponding to an approximately 3.5-fold increase in cell volume. Similar changes in the chondrocyte cell size were induced by latrunculin and cytochalasin D (Fig. 4A), suggesting that the observed adseverin-induced hypertrophy of chondrocytes depends on cytoskeleton rearrangement. Our data may explain, at least partially, the reported enlargement of chondrocytes during differentiation (Farnum et al., 2002) and identify adseverin as the first actin-binding protein shown to be involved in this process.

Figure 4. Adseverin-induced changes in chondrocyte morphology.

A – Increase is cell diameter of non-hypertrophic chondrocytes induced by rearrangement of actin cytoskeleton resulting from exposure to latrunculin and cytochalasin D, or from over-expression of adseverin. Cells transfected with eietrh pDsRed or pAdseverin-DsRed were grown in 3D-collagen gels. Fluorescent cells were photographed and cell diameter was measured in pixels. At least 100 cells per condition were analyzed. Experiment was repeated twice. B- Over-expression of adseverin in non-hypertrophic chondrocytes (NH) alters the morphology of cells grown in monolayers, inducing the polygonal phenotype characteristic for hypertorphic chondrocytes (Hyp). Cells expressing DsRed protein retain the fibroblastic morphology characteristic for dedifferentiating chondrocytes.

In addition to the demonstrated ability to induce an increase in chondrocyte cell volume when cells are cultured in the collagen gels, adseverin can cause dramatic changes in cell morphology. When non-hypertrophic chondrocytes expressing adseverin are grown in monolayers, they display the polygonal morphology characteristic of hypertrophic cells (Fig. 4B), in contrast to the fibroblast-like morphology of non-transfected cells, or of the cells over-expressing the control protein DsRed (Fig. 4B). The acquisition of the hypertrophic phenotype by the non-hypertrophic cells over-expressing adseverin supports the hypothesis on the interdependence of cell size/shape, cytoskeleton arrangement, and the program of chondrocyte differentiation.

4. Adseverin regulates chondrocyte proliferation and differentiation

Chondrocyte transition from the non-hypertrophic to hypertrophic stage is accompanied by a decline and eventual cessation of proliferation. To assess the effect of adseverin on proliferation rates of non-hypertrophic chondrocytes, we isolated transfected non-hypertrophic chondrocytes, expressing either Adseverin-DsRed fusion protein or DsRed, by fluorescent cytometry. Fluorescent cells were plated at a very low density (500 cells/cm²) to allow for the growth of individual colonies of transfected cells in the G418 selection medium. Non-hypertrophic cells expressing the DsRed protein proliferated rapidly, and formed extended colonies of fluorescent cells with some cells demonstrating the fibroblast-like morphology of dedifferentiated chondrocytes (Fig. 5A, right panel). On the contrary, the colonies of chondrocytes expressing Adseverin-DsRed grew very slowly with cells acquiring the hypertrophic chondrocyte-like polygonal shape and/or rounding up, likely due to the rearrangement of the actin cytoskeleton. (Fig. 5A, left panel)

Figure 5. Adseverin-induced accelerated chondrocyte differentiation is mediated by MAPK signaling.

A-Over-expression of adseverin in non-hypertrophic chondrocytes slows down proliferation in non-hypertrophic chondrocytes. FACS-sorted cells over-expressing either Adseverin-DsRed or DsRed control were plated at very low density, and the growth of individual colonies was analyzed by fluorescent microscopy in 7 day-old cultures. B - Over-expression of adseverin (Ads), but not of DsRed (Red), induces activation of ERK 1/2 and p38 signaling pathways in non-hypertrophic chondrocytes detected by Western blot. C – expression levels of adseverin, Ihh and collagen type X were compared in the absence or presence of the p38 inhibitor SB203590 (15 µM) and MEK1/2 inhibitor U0126 (5 µM) in non-hyeprtrophic chondrocytes induced to differentiate with ascorbic acid. Inhibition of p38 kinase (black bars) results in decreased expression of collagen type X (marker of late hypertrophy) but does not affect expression of adseverin and Ihh (markers of pre-hypertrophic stage), whereas inhibition of the MEK1/2-ERK1/2 pathway (grey bars) results in concurrent down-regulation of adseverin and Ihh. All data represent averages and SD from three independent experiments (*p < 0.05).

The decline in proliferation is associated with chondrocyte maturation during differentiation. To address whether the observed adseverin-induced limited growth in non-hypertrophic chondrocytes is associated with an accelerated differentiation towards hypertrophy, we analyzed the expression of Indian hedgehog (Ihh), a marker of pre-hypertrophic chondrocytes, and of collagen type X, specific for hypertrophic chondrocytes, by real-time PCR in non-hypertrophic chondrocytes expressing either Adseverin-DsRed or DsRed. FACS-sorted transfected cells were grown for 10-days to allow for differentiation. Hypertrophic chondrocytes isolated from the growth plate were used as a positive control (Table 1). Non-hypertrophic chondrocytes transfected with pDsRed as well as the non-transfected cells (data not shown) expressed extremely low levels of either Ihh or collagen type X after 10 days in culture. Over-expression of adseverin in non-hypertrophic chondrocytes induced expression of both genes. The most dramatic increase in expression was detected for Ihh (with ca. 25-fold increase in Ihh mRNA levels in the adseverin-expressing cells), while expression of type X collagen was increased but not to the extraordinary high levels of this protein characteristic to mature hypertrophic chondrocytes. These results indicate that transition from the immature to the pre-hypertrophic stage of chondrogenesis is more dependent on the cytoskeleton organization than the final chondrocyte maturation.

Table 1.

Adseverin-induced expression of the markers of chondrocytes differentiation in non-hypertrophic chondrocytes.

	Ihh	Collagen type X
NH-DsRed	48 +/−22	4 +/− 1
NH-Adseverin	1118 +/− 225	205 +/− 28
Hyp	358 +/− 198	7191 +/− 870

Transiently transfected non-hypertrophic chondrocytes were grown in the G418-selection medium for 10 days, sorted by fluorescent cytometry and allowed to differentiate for 5 days in the presence of ascorbic acid. Expression levels of Ihh and collagen type X were analyzed by the real-time PCR. Expression of the analyzed genes in non-hypertrophic chondrocytes, freshly liberated from the cartilage, has been used as basal levels. Data is normalized to beta-actin

5. Adseverin-induced chondrocyte differentiation is mediated by ERK1/2 and p38 kinases

Chondrocyte differentiation is in general regulated by the mitogen-activated protein (MAP) kinase signaling pathways. Both the ERK1/2 and the p38 kinases are known to regulate expression of Ihh in maturing chondrocytes (Haroon et al., 1999). To determine whether these pathways mediate the adseverin-induced expression of Ihh, we analyzed the levels of activated (phosphorylated) ERK1/2 and p38 in non-hypertrophic chondrocytes over-expressing adseverin. Cells were transfected with either pAdseverin-DsRed or pDsRed, allowed to grow in the G418-selection medium for 10 days and sorted by FACS to enrich the analyzed samples with transfected cells. Both the ERK1/2 and the p38 signaling pathways are activated by over-expression of adseverin, as detected by Western blot analysis (Fig. 5B). At the same time, no adseverin-induced changes were detected in the JNK signaling pathway (data not shown). These data identify ERK1/2 and p38 kinases as molecular mediators of the adseverin-induced accelerated chondrocyte differentiation, especially the transition into early hypertrophic stage characterized by expression of Ihh.

To determine the hierarchy and interdependence of adseverin-induced actin rearrangement and activation of MAP kinases, we analyzed the expression of adseverin, Ihh and collagen type X in differentiating chondrocytes exposed to various kinase inhibitors. Non-hypertrophic chondrocytes were grown for 5 days with ascorbic acid. Under these conditions, the cells start to differentiate into hypertrophic chondrocytes and initiate expression of Ihh, collagen type X, and adseverin, as determined by real-type PCR analysis (Fig. 5C, white bars). Inhibition of the p38 kinase with a specific inhibitor SB202190 significantly decreases expression of Ihh and collagen type X (Fig. 5B, black bars). These results are consistent with the previously published data on the requirement of the p38 kinase activity for chondrocyte maturation and collagen type X expression (You et al., 2001) as well as for the Ihh expression (Lai et al., 2005). At the same time, inhibition of the p38 kinase had little, if any, effect on expression of adseverin (Fig. 5C, black bars). In concert with the observed activation of p38 by adseverin expression (Fig.5B), these results imply that the p38 kinase signaling is a downstream target of the adseverin-induced rearrangements of actin cytoskeleton.

On the contrary, inhibition of ERKs by U0126, the inhibitor of the upstream MEK kinase, caused an approximately 60% concurrent decrease in expression of Ihh and adseverin (Fig. 5C, grey bars). These results indicate that adseverin expression in chondrocyte hypertrophy is regulated by MEK signaling, in addition to the demonstrated regulation by the PKC activity (Fig. 3). This is in agreement with the recent findings of Oh and co-authors (Oh et al., 2000) that ERK1/2-mediated PKC signaling regulates chondrocyte differentiation. Activation of ERK1/2 can lead to actin rearrangements (Sukezane et al., 2005), and our data identify adseverin as a candidate mediator of such rearrangements in chondrocytes. Interestingly, over-expression of adseverin in non-hypertrophic chondrocytes results in activation of ERK1/2 (Fig. 5B), suggesting a positive regulatory feedback that maintains the ERK1/2 activity in maturing chondrocytes.

In summary, we have observed that actin-binding protein adseverin is expressed in pre-hypertrophic and hypertrophic chondrocytes of the embryonic growth plate. Expression of adseverin in differentiating chondrocytes is controlled by the PKC-MEK signaling and leads to increased cell size, decreased chondrocyte proliferation and induction of the Ihh and collagen type X expression, indicative of chondrocyte differentiation. MAP kinases p38 and ERK1/2 mediate the adseverin-induced accelerated differentiation of non-hypertrophic chondrocytes.

DISCUSSION

The phenomenon of interdependence of cell shape, cytoskeletal organization and chondrogenic phenotype has been well established few decades ago (reviewed in (Cancedda et al., 1995)). A role for actin cytoskeleton in chondrocyte differentiation was implicated by the initial observations on the ability of spherical chondrocytes cultured in agarose gels to retain their chondrogenic phenotype but to lose it when cultured in monolayers or on fibronectin (Benya and Shaffer, 1982); (Glowacki et al., 1983)). Subsequent studies on disruption of actin microfilaments with cytochalasin D in chick limb bud mesenchymal cells in subconfluent monolayers which resulted in cell rounding, loss of actin cables and induction of chondrogenesis confirmed regulation of chondrogenesis by cytoskeleton (Zanetti and Solursh, 1984). The assembly and disassembly of the actin cytoskeleton in response to extracellular matrix is controlled by small Ras-related GTP-binding proteins RhoA and Rac1 and Cdc42. (Hall, 2005). All of these have been recently implicated as regulators of chondrocyte differentiation ((Woods et al., 2005); (Woods et al., 2005)). Nevertheless, the molecular mechanisms underlying the regulation of chondrocyte maturation by actin cytoskeleton are not clearly understood. In particular, surprisingly little is known about the chondrocyte-specific actin-binding proteins that regulate cytoskeletal rearrangement, despite the existence of more than 60 classes of such proteins discovered in eukaryotic cells (reviewed in (Tojima and Ito, 2004). Up to date, only filamins A and B have been shown to affect endochondral ossification. Human patients with mutated filamins are characterized by defects restricted to vertebral segmentation, joint formation and endochondral ossification, despite the ubiquitous expression of these proteins ((Krakow et al., 2004); (Robertson et al., 2003)). These studies further reinforced the link between actin cytoskeleton and chondrogenesis, however the chondrocyte-specific mechanism responsible for the effect remained elusive.

Previously, we identified an actin-binding protein of gelsolin family as product of one of the genes up-regulated during chondrocyte differentiation (Nurminskaya and Linsenmayer, 1996) Here we identified this protein as adseverin. The expression pattern of several proteins of gelsolin family has been analyzed in mouse development by in situ hybridization. This study detected only adseverin, but not gelsolin or capG protein, in embryonic endochondral bones (Arai and Kwiatkowski, 1999). We analyzed expression pattern of gelsolin and adseverin in cultured chicken chondrocytes at different stages of maturation, and observed that gelsolin is expressed at almost identical levels in non-hypertrophic and hypertrophic chondrocytes, while adseverin is dramatically up-regulated during progression towards hypertrophy. The restricted pattern of adseverin expression in prehypertrophic and hypertrophic zones of the embryonic growth plate was confirmed using Northern blot analysis, real-time PCR, and in situ hybridization. Thus, adseverin represents an actin-binding protein of gelsolin family specifically up-regulated during chondrocyte hypertrophy.

Although adseverin is thought to regulate exocytosis in chromaffin cells (Trifaro et al., 2002), its physiological role in developing bones and in other tissues without significant secretory activity remains unknown. Our data demonstrate that adseverin may contribute to chondrocyte enlargement associated with hypertrophy (Barreto and Wilsman, 1994), since over-expression of this protein in chondrocytes results in an approximately 3.5-fold increase in cell volume. The forced expression of adseverin in non-hypertrophic chondrocytes also induces a dramatic change in cell morphology, with transfected cells grown in monolayers acquiring the polygonal cell shape characteristic for hypertorphic chondrocytes. These changes are accompanied by adseverin-induced actin depolymerization, enrichment with actin monomers, and cytoskeletal rearrangements. In addition, premature expression of adseverin in non-hypertrophic chondrocytes results in slower cell proliferation and acceleration of chondrocyte maturation, as evidenced by the up-regulation of specific chondrogenic markers Ihh and collagen type X. Adseverin-induced increase in Ihh expression in non-hypertrophic chondrocytes is more pronounced than that of type X collagen, when compared to the endogenous levels of these proteins in hypertrophic cells. This suggests that adseverin regulates the intermediate stages of chondrocyte differentiation (transition from pre- to early hypertrophy) rather than the final stage of maturation.

The expression of adseverin itself in chondrocytes appears to be regulated by the PKC-MEK signaling. Inhibition of PKC in differentiating chondrocytes by bisindolylmaleimide I results in morphological dedifferentiation and cessation of transcription of adseverin and of the downstream makers of hypertrophy such as Ihh and collagen type X. Inhibition of the MEK in these cells with U0126 resulted in a significant (approximately 50%) decrease in expression of the early hypertrophic genes, such as adseverin and Ihh, thus suggesting a positive regulation of chondrocyte maturation by this pathway. Earlier studies have indicated that PKC signaling is required for chondrogenesis ((Chang et al., 1998); (Oh et al., 2000); (Yoon et al.,2002)). The precise role of the MEK-ERK cascade in the regulation of chondrocyte differentiation remains controversial. Several studies have demonstrated positive regulation of in vitro chondrogenesis by this pathway (Murakami et al, 2004); (Nakajima et al., 2004)) while others describe this cascade as a negative modulator of cartilage differentiation ((Chang et al., 1998); (Bobick and Kulyk, 2004); (Seghatoleslami et al., 2003)). The discrepancy is in large due to the employed kinase inhibitors. The two most commonly used inhibitors of the MEK-ERK cascade - U0126 and PD98059 –likely do not have completely equivalent target specificities (Bobick and Kulyk, 2004) and confer opposite effects on chondrogenesis in a dose-dependent manner (Seghatoleslami et al., 2003). In our studies, we used U0126 which is known to inhibit chondrocyte differentiation ((Murakami et al., 2000); (Stephens et al., 2004); (Nakajima et al., 2004)); this inhibition resulted in down-regulation of adseverin and Ihh. Thus, our analysis demonstrated that treatment with the PKC-MEK-ERK pathway inhibitors that impede chondrocyte differentiation results in down-regulation of adseverin and of the downstream hypertrophy markers Ihh and (in the case of PKC inhibitor) collagen type X. These data suggest the role for adseverin as a mediator of pro-differentiation activity of the PKC-MEK-ERK pathway in chondrocytes.

Up-regulation of adseverin results in changes in actin cytoskeleton, and in expression of the components of the chondrocyte differentiation program such as Ihh and collagen type X. These events are probably linked by the p38 kinase pathway. We demonstrated that elevated levels of adseverin activate p38 kinase in chondrocytes. The p38 kinase is an essential regulator of chondrocyte differentiation and is required for Ihh and collagen type X expression ((You et al., 2001); (Haroon et al., 1999); (Nakajima et al., 2004)). Modulations of p38 kinase signaling by the actin cytoskeleton have been proposed for articular chondrocytes ((Kim et al., 2003); (Szondy et al., 2003)). In addition, the p38 kinase activity was implicated to mediate chondrocyte maturation induced by small GTPases Rac1 and Cdc42, which affect actin cytoskeleton, in the chondrogenic cell line ATDC5 (Woods et al., 2005). Taken together, all these data suggest that the p38 kinase pathway may represent the downstream target of adseverin, regulated through the changes in actin cytoskeleton.

Our data also imply that adseverin regulates the MEK-ERK1/2 signaling: over-expression of adseverin in chondrocytes results in activation of ERK1/2. At the same time, the MEK-ERK1/2 signaling is required for high levels of adseverin expression, as discussed above. These observations suggest two signaling events mediated by ERK1/2 contributing to chondrocyte maturation. The first event, which requires PKC (Chang et al., 1998), leads to initiation of chondrocyte progression to hypertrophy and induction of pre-hypertrophic markers such as adseverin and Ihh. Up-regulation of adseverin leads to actin rearrangement and to the second activation of ERK1/2, which probably accelerates chondrocyte hypertrophy. Progression of chondrocyte maturation requires a coordinate regulation of small GTPases Rho and Rac1/Cdc42, which antagonistically regulate chondrocyte proliferation, hypertrophy and apoptosis (Woods et al., 2005). Intriguingly, the ERK signaling can coordinately regulate activity of Rac1 and RhoA in the required fashion, as has been demonstrated in tumor cells (Vial et al., 2003). Therefore, we propose a model (Fig. 6) in which activation of PKC in non-hypertrophic chondrocytes induces the genes of the early differentiation program, including the actin-binding protein adseverin. Elevated levels of adseverin cause the reorganization of actin cytoskeleton leading to activation of the signaling through the p38 and ERK1/2 kinases. (Alternatively, adseverin may stabilize or amplify the activity of MEK-ERK1/2 cascade during chondrocyte differentiation but another, yet unknown, mechanism). Activation of the MAPK signaling promotes chondrocyte hypertrophy, including the morphological changes (e.g. changes in cell morphology and increase in cell size) and the gene expression program (i.e. expression of markers of chondrocyte maturation).

Figure 6. Scheme of the role of adseverin in regulation of cytoskeleton-dependent chondrocyte differentiation.

Activation of PKC signaling in non-hypertrophic chondrocytes induces adseverin expression in a MEK-dependent manner. Expression of adseverin causes rearrangements of actin cytoskeleton leading to chondrocyte maturation evident by cell enlargement and changes in cell morphology, and also by induced expression of Ihh and collagen type X. The changes in gene expression are regulated by adseverin-induced activation of the ERK1/2 and p38 kinase signaling pathways. Additionally, MEK-ERK1/2 activity is required to maintain adseverin expression suggesting a regulatory positive feedback between these proteins.

Further studies of the physiological functions of adseverin in embryonic endochondral bone formation will require the genetic models. Compensation between adseverin and the other members of gelsolin protein family expressed in cartilage, such as the gelsolin, may complicate the analysis of the individual proteins. For example, gelsolin null mice develop normally (Silacci et al., 2004). Generation of double knockout models will further our understanding on the roles of gelsolin and adseverin in the molecular mechanisms controlling bone formation.

ABBREVIATIONS

NH

non-hypertrophic chondrocytes

HYP

hypertrophic chondrocytes

Ihh

Indian hedgehog