Bcl-2-associated athanogene-1 (BAG-1): A transcriptional regulator mediating chondrocyte survival and differentiation during endochondral ossification

Bone 42 (2008) 113 – 128 www.elsevier.com/locate/bone

Bcl-2-associated athanogene-1 (BAG-1): A transcriptional regulator mediating chondrocyte survival and differentiation during endochondral ossification Rahul S. Tare a , Paul A. Townsend b , Graham K. Packham c , Stefanie Inglis a , Richard O.C. Oreffo a,⁎ a

Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK b Human Genetics Division, University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, UK c Cancer Sciences Division, University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, UK Received 7 March 2007; revised 2 August 2007; accepted 6 August 2007 Available online 4 September 2007

Abstract BAG-1, an anti-apoptotic protein, was identified by its ability to bind to BCL-2, HSP70-family molecular chaperones and nuclear hormone receptor family members. Two BAG-1 isoforms, BAG-1L (50 kDa) and BAG-1S (32 kDa) were identified in mouse cells and BAG-1 expression was reported in murine growth plate and articular chondrocytes. The present study aimed to elucidate the role of BAG-1 in the regulation of molecular mechanisms governing chondrocyte differentiation and turnover during endochondral ossification. In long bones of skeletally immature mice, we observed expression of BAG-1 in the perichondrium, osteoblasts, osteocytes in the bone shaft, bone marrow, growth plate and articular chondrocytes. Monolayer cultures of murine chondrocytic ATDC5 cells, which exhibited robust expression of both BAG-1 isoforms and the Bag-1 transcript, were utilized as an in vitro model to delineate the roles of BAG-1. Overexpression of BAG-1L in ATDC5 cells resulted in downregulation of Col2a1 expression, a gene characteristically downregulated at the onset of hypertrophy, and an increase in transcription of Runx-2 and Alkaline phosphatase, genes normally expressed at the onset of chondrocyte hypertrophy and cartilage mineralization in the process of endochondral ossification. We also demonstrated the anti-apoptotic role of BAG-1 in chondrocytes as overexpression of BAG-1 protected ATDC5 cells, which were subjected to heat-shock at 48 °C for 30 min, against heat-shock-induced apoptosis. Overexpression of the SOX-9 protein in ATDC5 cells resulted in increased Bag-1 gene expression. To further investigate the regulation of Bag-1 gene expression by SOX-9, CHO cells were co-transfected with the human Bag-1 gene promoter–Luciferase reporter construct and the human pSox-9 expression vector. Activity of the Bag-1 promoter was significantly enhanced by the SOX-9 protein. In conclusion, a novel finding of this study is the role of BAG-1 as a transcriptional regulator of genes involved in chondrocyte hypertrophy and cartilage mineralization during the process of endochondral ossification. Additionally, we have demonstrated for the first time the regulation of Bag-1 gene expression by SOX-9 and the anti-apoptotic role of BAG-1 in chondrocytic cells. Modulation of Bag-1 expression can therefore mediate chondrocyte differentiation and turnover, and offer further insight into the molecular regulation of endochondral ossification. © 2007 Elsevier Inc. All rights reserved. Keywords: ATDC5; Chondrocyte differentiation; Endochondral ossification; Runx-2; Alkaline phosphatase

Introduction

⁎ Corresponding author. Fax: +44 2380 796141. E-mail address: [email protected] (R.O.C. Oreffo). URL: http://www.mesenchymalstemcells.org.uk (R.O.C. Oreffo). 8756-3282/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2007.08.032

Long bone formation occurs in the growth plate cartilage by the regulated process of endochondral ossification [1]. As a part of this process, growth plate chondrocytes undergo discrete differentiation stages whereby these cells sequentially proliferate, mature and undergo terminal differentiation or hypertrophy. Once fully differentiated, hypertrophic chondrocytes participate

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in mineralization of the cartilaginous matrix and undergo cell death. As early as 1973, dying hypertrophic chondrocytes have served to demonstrate a mechanism of cell death (non lysosomal degradation) morphologically distinct from the widely studied modes of physiological cell death namely, apoptosis and autophagic cell death [2]. In addition to hypertrophic chondrocytes, some chondrocytes of the proliferative and upper hypertrophic zones of the growth plate have been demonstrated to undergo cell death [3]. Previous studies have shown the coexistence of two morphologically distinct types of chondrocytes (light and dark) in the proliferative and hypertrophic zones of the growth plate [4,5]. Ultrastructural characterisation of the modes of cell death adopted by light and dark chondrocytes revealed two morphologically distinct mechanisms different from classical apoptosis [3,6]. The term ‘chondroptosis’ has been introduced in literature to describe the mechanism of cell death adopted by dark chondrocytes [7]. Another study has suggested that hypertrophic chondrocytes die through the induction of autophagy, which causes sensitisation of these terminally differentiated cells to apoptogens in the local microenvironment [8]. Cartilage formation is a complex process and intricate molecular mechanisms regulate differentiation of chondrocytes in the growth plate. Abundant Sox-9 expression is observed during mouse embryonic development in mesenchymal condensations before overt chondrocyte differentiation, and expression of this transcription factor is found to persist during the subsequent stages of cartilage deposition [9]. Typically, chondrocytes express a set of genes encoding cartilage-specific extracellular matrix components such as Type II collagen (encoded by the Col2a1 gene), Type IX collagen, Type XI collagen and aggrecan. SOX-9 binds directly to the Col2a1 enhancer at a site which is essential for Col2a1 expression in chondrogenic cells [10]. Induction of Col2a1 expression occurs in the growth plate with the change in cellular phenotype from prechondrogenic cells to proliferating chondrocytes [11], while expression of Col2a1 and Sox-9 declines in hypertrophic chondrocytes [12,13]. The induction of Col10a1 expression stimulates the change in cellular phenotype from proliferating/prehypertrophic chondrocytes to hypertrophic chondrocytes, and expression of Type X collagen is restricted to hypertrophic chondrocytes [14]. Bcl-2, an anti-apoptotic molecule, is expressed in the growth plate in late proliferative and prehypertrophic chondrocytes, while expression decreases in hypertrophic chondrocytes [15]. Apart from its role in preventing apoptosis, BCL-2 is involved in a number of pathways vital in the maintenance of a stable chondrocyte phenotype. Suppression of Bcl-2 expression results in downregulation of Sox-9, Col2a1 and Aggrecan expression [16,17]. Recently, a protein termed Bcl-2-associated athanogene-1 (BAG-1) has been demonstrated to be expressed in mouse growth plate and articular chondrocytes [18]. The study demonstrated changes in the pattern of BAG-1 expression by growth plate chondrocytes in mice aged 6 weeks, 6 months and 18 months, and expression of BAG-1 in chondrocytes decreased with age. BAG-1 is a ubiquitously expressed protein, originally identified by its ability to bind to and enhance the anti-apoptotic

activity of the BCL-2 protein [19], and interact with the members of the nuclear hormone receptor family [20]. Six genes in mammalian cells encode the BAG-family of proteins, namely, Bag-1 (encoding its various isoforms including RAP46/ HAP46, HAP50), Bag-2, Bag-3 (CAIR-1, BIS), Bag-4 (SODD), Bag-5 and Bag-6 (SCYTHE, BAT3) [21]. Proteins of the BAG-family are characterised by an evolutionarily conserved BAG domain that allows them to bind and modulate the activity of the HSP70-family molecular chaperones [22–25]. Multiple BAG-1 isoforms are expressed in human and murine cells, generated as a result of alternate translation initiation sites in a single mRNA transcript [26]. In addition to the cytosolic 32 kDa and 36 kDa BAG-1S isoforms identified in murine and human cells respectively, the existence of a larger 50 kDa BAG-1L isoform, containing the N-terminal nuclear localisation sequence (NLS), has been confirmed in the nucleus [26]. The BAG-1S isoform, however, has been reported to translocate from the cytoplasm to the nucleus in cells subjected to heat-shock [27]. The BAG-1L isoform is translated from a non canonical CUG codon, while the BAG-1S isoform is translated from a downstream AUG codon [26]. BAG-1S translation is also thought to be partially mediated via an internal ribosome entry segment [28]. In addition to the BAG-1L and BAG-1S isoforms, human cells express a 46 kDa BAG-1M isoform (RAP46/HAP46), which arises as a result of translation initiation at yet another site involving an AUG codon and this isoform partitions itself between the nucleus and cytoplasm [29]. Studies on the developing limb bud during mouse embryogenesis reveal that Bag-1 is initially expressed throughout the mesenchyme of the developing limb bud between E10.5 and E11.5 [30]. At E14.5, when digit formation is nearly complete, Bag-1 expression is not detected in the cartilaginous anlagen and is downregulated in the mesenchymal tissues programmed to undergo apoptosis i.e. in the interdigital spaces [30]. Bag-1

Table 1 qPCR primer sequences and their amplicon sizes Gene

Primer pairs

Amplicon (bp)

β-Actin

F: 5′ TTG CTG ACA GGA TGC AGA AG 3′ R: 5′ GTA CTT GCG CTC AGG AGG AG 3′ F: 5′ GAG GCC ACG GAA CAG ACT CA 3′ R: 5′ CAG CGC CTT GAA GAT AGC ATT 3′ F: 5′ CGA GTG GAA GAG CGG AGA CT 3′ R: 5′ AAC TTT CAT GGC GTC CAA GGT 3′ F: 5′ TCG CAG AGA TGT CCA GTC AG 3′ R: 5′ CCT GAA GAG TTC CTC CAC CA 3′ F: 5′ ACG GCA CGC CTA CGA TGT 3′ R: 5′ CCA TGA TTG CAC TCC CTG AA 3′ F: 5′ CAG ACG GAG GAA ATG GAA AC 3′ R: 5′ GCT GTG GGG TAA CAA GAA GG 3′ F: 5′ CCA CCA CTC ACT ACC ACA CG 3′ R: 5′ CAC TCT GGC TTT GGG AAG AG 3′ F: 5′ CTG ACT GAC CCT TCG CTC TC 3′ R: 5′ CCA GCA AGA AGA AGC CTT TG 3′

85

Sox-9 Col2a1 Bcl-2 Col10a1 Bag-1 Runx-2 Alkaline phosphatase

50 66 82 77 84 63 82

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knock-out mice are characterized by significant cell death in the embryonic liver, defective haematopoiesis and severe defects in the differentiation and survival of neuronal cells, resulting in their death between E12.5 and E13.5 [31].

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The role of BAG-1 in postnatal endochondral bone development, however, remains to be elucidated. We hypothesize that BAG-1 plays an important role in the process of endochondral bone development. The present study set out to

Fig. 1. Regions of BAG-1 expression (b – growth plate, c – articular cartilage, d – perichondrium, e – bone shaft and marrow) highlighted in the longitudinal section of distal end of femur from 10-week-old BDF-1 mouse (A). BAG-1 was immunolocalised to growth plate chondrocytes (B), articular chondrocytes (C), perichondrium (arrowhead in panel D) and palisade of osteoblasts (block arrows in panel D). Expression was detected in osteocytes (arrowheads in panel E) of bone shaft and in the bone marrow (block arrow in panel E). Western blot demonstrating expression of BAG-1L and BAG-1S isoforms in day 12 murine bone marrow cell cultures under basal conditions (E). Control sections exhibiting absence of immunostaining when the primary antibody was blocked by preincubating with the BAG-1L and BAG-1S recombinant proteins (F–H). Scale bar: 100 μm.

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Fig. 2. Distribution of BAG-1 in growth plates of mice aged 3 (A) and 10 weeks (B). Growth plates demonstrated well-defined regions of chondrocyte differentiation namely, reserve (RZ), proliferative (PZ), upper hypertrophic/maturing/prehypertrophic (UHZ) and hypertrophic (HZ) zones. At 3 weeks, BAG-1 expression in the growth plate was predominantly observed in chondrocytes of the reserve and upper hypertrophic zones, and in few hypertrophic chondrocytes (A). At 10 weeks, BAG-1 was expressed by upper hypertrophic and hypertrophic zone chondrocytes (B). Scale bar: 100 μm.

delineate the role of BAG-1 in the regulation of molecular mechanisms governing chondrocyte differentiation and turnover during endochondral ossification.

Cells were grown to confluence for a period of 12 days under basal culture conditions i.e. in α-MEM supplemented with 10% FCS, and harvested for analysis of BAG-1 protein expression by Western blotting.

Sample processing and paraffin embedding Materials and methods Animals used in the study The murine BDF-1 strain is the F1 generation hybrid progeny of C57 B1/6/ Ola/Hsd (black) and DBA/2/Ola/Hsd (grey) strains of mice. The mice were purchased from a registered supplier and bred in the University of Southampton Animal Facility. Animals were housed in appropriate environments maintained at 22 °C with 12-h/12-h light/dark cycles, had free access to water and fed ad libitum. All animal experimentation was performed under license from the Home Office in accordance with the Animals Act (1986).

Cell culture ATDC5 monolayer cell culture ATDC5 cells were grown as monolayer cultures in DMEM, supplemented with 5% fetal calf serum (FCS) and 1× ITS (insulin – 10 μg/ml, transferrin – 5.5 μg/ml, selenium – 5 ng/ml) premix (Sigma-Aldrich Co., Ltd.), over a period of 28 days at 37 °C in a humidified environment of 5% CO2. The cells were harvested at days 2, 7, 14, 21 and 28 for analysis of gene and protein expression by Real-time quantitative PCR (qPCR) and Western blotting, respectively. Murine bone marrow cell culture Primary cultures of bone marrow-derived cells from two 10-week-old BDF-1 mice were established in accordance with the protocol described previously [32].

Femora and tibiae, dissected from male and female BDF-1 mice aged 3 and 10 weeks (an average number of three mice for each age group) were fixed in 4% phosphate-buffered paraformaldehyde overnight. The samples were then processed through graded alcohols and chloroform, and embedded in paraffin wax. Sequential sections were cut at 5 μm on the microtome for histological analysis.

Immunohistochemistry The rabbit α-rodent BAG-1 polyclonal antibody utilised in this study was capable of detecting both BAG-1 isoforms, and was raised against a chimaeric protein containing the mouse BAG-1 protein fused to the C-terminus of GST [26]. After quenching endogenous peroxidase activity with 3% H2O2 and blocking with 1% bovine serum albumin in phosphate-buffered saline, sections were incubated with the α-BAG-1 antibody (1 in 500 dilution) at 4 °C overnight, followed by an hour's incubation with the goat anti-rabbit IgG-biotinylated secondary antibody (Dako UK Ltd.). Visualization of the immune complex involved the avidin–biotin method linked to peroxidase and 3-amino-9ethylcarbazole (AEC), resulting in a reddish brown reaction product. Controls involving omission of the primary antibody were included in all immunohistochemistry procedures. An additional control involved blocking of the primary antibody by preincubating with the murine BAG-1L and BAG-1S recombinant proteins. No staining was observed in the control sections. All sections were counterstained with Alcian blue.

Fig. 3. Time course of murine chondrocytic ATDC5 cells cultured in a monolayer in presence of insulin, transferrin and selenium over a 28-day period demonstrating sub-confluent cultures at day 2, confluent cultures at day 7, formation of cartilaginous nodules at day 14 and well-defined cartilaginous nodules at days 21 and 28 (A). Expression of the BAG-1 protein and its mRNA transcript was analysed along with the expression of SOX-9, Type II collagen and BCL-2 proteins, and their mRNA transcripts during the course of ATDC5 cell differentiation by Western blotting (B) and qPCR (C), respectively. Relative expression levels for each gene were normalised to the expression of the β-Actin gene. For each gene, the group with the highest expression was assigned a value of 1 and expression levels in the remaining groups were determined relative to the group exhibiting the highest expression. Fold relative expression levels were expressed as mean ± SD (n = 3) for plotting as bar graphs. Both BAG-1 isoforms and the Bag-1 transcript were expressed throughout the course of ATDC5 differentiation. A band of ∼46 kDa (denoted by a star), in addition to the 50 kDa BAG-1L and 32 kDa BAG-1S bands, was observed in the Western blot for BAG-1 expression in the ATDC5 time course. Scale bar: 100 μm.

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Heat-shock treatment of ATDC5 cells overexpressing BAG-1 The murine pEGFP–Bag-1L expression construct was a generous gift from Dr Stefan Wiese, University of Würzburg, Germany. ATDC5 cells, cultured in

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6-well plates to about 40% confluency, were transfected with 1 μg/well of either the murine pEGFP–Bag-1L expression construct or the pEGFP empty vector using the FuGENE 6 transfection reagent in accordance with the manufacturer's protocol. Localisation of GFP and GFP–BAG-1 proteins in

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ATDC5 cells, nuclei of which were stained with DAPI (Invitrogen), was observed using the Zeiss Axiovert 200 microscope. Western blot analysis was performed to establish overexpression of the BAG-1 proteins in ATDC5 cells as a

result of transfection with the pEGFP–Bag-1L expression construct. Approximately 40 h post-transfection, the cells were subjected to heat-shock by incubating the 6-well plates for 30 min at 48 °C in an in situ hybridisation oven,

R.S. Tare et al. / Bone 42 (2008) 113–128 following which the cells were incubated overnight at 37 °C, 5% CO2 in a humidified atmosphere. Apoptotic cells were detected using the APOPercentage™ Apoptosis assay (Biocolor Ltd. UK), a unidirectional dye-uptake bioassay that only marked apoptotic cells and not necrotic cells. Once the dye had accumulated in the apoptotic cells, any unbound dye was washed off with PBS and the cells fixed in 4% phosphate-buffered paraformaldehyde. The total number of cells and the number of apoptotic cells in each group (3 wells per group) was determined in five random fields per group using the MATLAB (v. 7.2 Image Processing Toolbox) and the GSA image analysis software, respectively.

Transient transfections with pBag-1L and pSox-9 expression constructs The murine pBag-1L expression construct was a generous gift from Dr Stefan Wiese, University of Würzburg, Germany. The coding sequence for the mouse BAG-1L protein was cloned into the pcDNA3.1 vector with two N-terminal HA tags. The human pSox-9 expression construct was a generous gift from Prof. Tim Hardingham, The University of Manchester, UK. The coding sequence for the human SOX-9 protein was cloned into the pcDNA3.1 vector with an N-terminal FLAG epitope. ATDC5 cells, cultured in 100 mm petriplates to about 40% confluency, were transfected with either the pBag-1L or pSox-9 expression constructs at concentrations of 1 μg and 2 μg/100 mm plate, utilising the FuGENE 6 transfection agent (ROCHE Diagnostics) in accordance with the manufacturer's protocol. ATDC5 cells transfected with the pcDNA3.1 vector (2 μg/100 mm plate) served as the empty vector (EV) control. Untransfected ATDC5 cells, grown for the same length of time as their transfected counterparts, served as an additional control (Ctrl.) for determining the basal levels of genes and proteins of interest. 24 h post-transfection, cells from three 100-mm plates of each group were harvested for RNA extraction, while cells from the fourth petriplate of that group were extracted in RIPA (Radio Immunoprecipitation) buffer for Western blotting.

Co-transfections with the pBag-1–Luciferase promoter–reporter construct and pSox-9 expression vector The human Bag-1 gene promoter (893 bp) construct with the Luciferase reporter (pBag-1–Luc) was a generous gift from Dr Xialong Yang and Dr ShouChing Tang, Memorial University of Newfoundland, Canada. Cloning and characterisation of this promoter have been previously described [33]. CHO cells were used for the purposes of co-transfection as it was not possible to detect endogenous SOX-9 and BAG-1 expression in these cells with the α-SOX-9 and α-BAG-1 antibodies used in the present study. To date, we are not aware of any published literature on SOX-9 and BAG-1 expression in these cells that contradicts our observations. Thus, CHO cells served as a neutral platform to study the effects of the overexpressed SOX-9 protein on the Bag-1 promoter. CHO cells, cultured in 6-well plates to about 40% confluency, were cotransfected with the pBag-1–Luc promoter–reporter construct (1 μg/well) and varying concentrations (200 ng, 400 ng, 600 ng, 800 ng/well) of the human pSox-9 expression construct or 400 ng/well pcDNA3.1 (empty vector control) using the FuGENE 6 transfection reagent in accordance with the manufacturer's protocol. As the human Bag-1 gene promoter was cloned into the pGL3-Basic vector, CHO cells grown to about 40% confluence in another 6-well plate were

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co-transfected with the promoterless pGL3-Basic vector (1 μg/well) and pSox-9 (400 ng/well). To monitor transfection efficiency, the cells were co-transfected with 25 ng/well of the promoterless Renilla Luciferase reporter construct. The cells were harvested 24 h post-transfection for the Dual-Luciferase assay (Promega).

Dual-Luciferase reporter assay Cells from each well were lysed in 0.2 ml 1× Passive Lysis Buffer (Promega) for 15 min. at room temperature. Firefly Luciferase activity was quantitated on a luminometer using a 10-s read time immediately after the addition of 5 μl cell lysate to 50 μl Luciferase substrate. Renilla Luciferase activity for each sample was then measured by adding 50 μl Stop & Glo reagent. Readings for Firefly Luciferase were divided by those for Renilla Luciferase for each sample, and the average values for each group (mean of 6 samples per group) were plotted as relative light units ± SD.

RNA extraction and qPCR analysis Total RNA was extracted from the ATDC5 cells using the TRIzol reagent (Invitrogen). The extracted RNA was subjected to DNAse treatment (DNA-free RNA kit, ZYMO Research) and reverse transcribed using the Super-Script Firststrand synthesis system for RT–PCR (Invitrogen). Real-time qPCR was performed using the 7500 Real Time PCR system from Applied Biosystems for analysing expression of β-Actin, Sox-9, Col2a1, Bcl-2, Col10a1, Runx-2, Alkaline phosphatase and Bag-1 genes. Primer sequences for these genes can be found in Table 1. As we used the SYBR Green method, the primer sequences for the genes were validated by dissociation curve/melt curve analysis. We also validated that the efficiencies of amplification for the β-Actin primers and primers for the genes of interest were approximately equal. The comparative CT method was employed for quantitation of gene expression. β-Actin served as the housekeeping gene, and relative expression levels for the genes of interest were normalised to β-Actin expression. For each gene, the group with the highest expression was assigned a value of 1 and expression levels in the remaining groups were determined relative to the group exhibiting the highest expression. Fold relative expression levels were expressed as mean ± SD (n = 3) for plotting as bar graphs.

Western blotting ATDC5 cell lysates were prepared by extraction in RIPA buffer containing the protease inhibitor cocktail (ROCHE Diagnostics), and protein concentrations were determined using the Bio-Rad protein assay, based on the Bradford dyebinding procedure. Protein samples (20 μg) were resolved in denaturing 10% polyacrylamide gels containing 10% SDS, and electroblotted onto nitrocellulose membranes. Membranes were blocked in a 5% solution of non fat powdered milk in PBS–0.01% Tween-20, and probed with the α-Actin antibody (rabbit polyclonal, 1 in 2000 dilution, Sigma-Aldrich Co., Ltd.), α-SOX-9 antibody (rabbit polyclonal, 1 in 500 dilution, Chemicon Europe, Ltd.), α-Type II collagen antibody (rabbit polyclonal, 1 in 500 dilution, Calbiochem), α-BCL-2 antibody (mouse monoclonal, 1 in 200 dilution, BD Biosciences), α-BAG-1 antibody (rabbit polyclonal, 1 in 500 dilution), α-HA antibody (rat monoclonal, 1 in 2000 dilution, ROCHE Diagnostics) and the α-FLAG antibody (mouse monoclonal, 1

Fig. 4. Expression of GFP–BAG-1L (nuclear) and -S (nuclear as well as cytosolic) isoforms in ATDC5 cells (subjected to heat-shock at 48°C for 30 minutes) 48 hours post-transfection with 1 μg/well pEGFP–Bag-1L expression construct (A). Cell nuclei were stained with DAPI (B) and merged image illustrated in panel C. Expression of GFP alone was observed in the cell nuclei (stained with DAPI) and cytoplasm of ATDC5 cells transfected with 1 μg/well pEGFP empty vector (D–F). Overexpression of the BAG-1 isoforms in ATDC5 cells up until 48 hours post-transfection was demonstrated by way of higher intensity of bands representing BAG-1L and BAG-1S proteins in lysates of ATDC5 cells transfected with 1 μg/well of the pEGFP–Bag-1L expression construct compared to the pEGFP empty vector (EV) control (G). Low magnification views of various groups of ATDC5 cells stained with the APOPercentage dye are shown in images labeled H, J, L and O. Corresponding high magnification views of these images are shown in panels I, K, M and P respectively, where apoptotic cells are stained pink with the APOPercentage dye. Images N and Q are high magnification views of M and P respectively. A negligible number of apoptotic cells were observed in cultures of ATDC5 cells that were transfected with either the pEGFP empty vector (H and I) or the pEGFP–Bag-1L expression construct (J and K), but were not subjected to heatshock. The level of heat-shock-induced apoptosis in ATDC5 cells overexpressing BAG-1 (O, P and Q) was significantly lower in comparison to the degree of apoptosis as a result of heat-shock in ATDC5 cells transfected with the pEGFP empty vector (L–N). The level of apoptosis in each group (3 wells per group) was determined by averaging the cell counts for stained cells in five random fields per group using the GSA image analysis software. Results were expressed as mean ± SD for plotting in the bar graph (R). ⁎⁎⁎P b 0.001. Scale bar: 20 μm in panels A–F; 100 μm in panels H–Q.

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R.S. Tare et al. / Bone 42 (2008) 113–128 in 2000 dilution, Sigma-Aldrich Co., Ltd.). Following incubation with the relevant HRP-conjugated secondary antibodies, the signal was detected using the Western blotting Luminol Reagent (Santa Cruz Biotechnology, Inc.).

Statistics Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey–Kramer Multiple Comparisons post-test using the GraphPad Instant Software (GraphPad Software Inc., San Diego, CA, USA). As the qPCR results were not log-transformed, ANOVA was performed on the raw data to test the significance of differences.

Results Distribution of BAG-1 in long bones of normal BDF-1 mice Regions of BAG-1 expression (b – growth plate, c – articular cartilage, d – perichondrium, e – bone shaft and marrow) highlighted in the longitudinal section of the distal end of femur from a 10-week-old BDF-1 mouse (Fig. 1A). The BAG-1 protein was immunolocalised in growth plate chondrocytes (Fig. 1B), articular chondrocytes (Fig. 1C), the perichondrium (arrowhead in Fig. 1D) and the palisade of osteoblasts (block arrows in Fig. 1D). Expression of the BAG-1 protein was maintained in osteocytes (arrowheads in Fig. 1E) of the bone shaft and intense staining was observed in the bone marrow (block arrow in Fig. 1E). To confirm our results for BAG-1 immunostaining in the marrow, bone marrow cells isolated from 10-week-old BDF1 mice and cultured in basal conditions for 12 days were examined for BAG-1 expression. Both BAG-1-L (50 kDa) and BAG-1-S (32 kDa) isoforms were detected in these cultures by Western blotting (inset in Fig. 1E). These cultures were also found to express Alkaline phosphatase (data not shown). No staining was observed in the control sections, notably in the chondrocytes, osteogenic cells and bone marrow, when the rabbit α-mouse BAG-1 antibody was blocked by preincubating with the murine BAG-1L and BAG-1S recombinant proteins (Figs. 1F–H). Expression of BAG-1 by growth plate and articular chondrocytes in BDF-1 mice (a hybrid strain) mirrored the results of a previous study in C57 B1/6/Ola/Hsd mice (a regular strain) by Kinkel and co-workers [18], suggesting that a normal pattern of BAG-1 expression was maintained in the BDF-1 hybrid mice. BDF-1 mice were therefore utilised in the present study to demonstrate the distribution of BAG-1 in long bones, as these mice were comparable to a normal/regular strain of mice. Expression of BAG-1 in the perichondrium, bone marrow and osteogenic cells demonstrated by the present study has not been reported previously.

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Immunolocalization of BAG-1 in growth plates of skeletally immature mice Rodents are characterised by rapid rates of endochondral bone development until skeletal maturity, which is achieved typically by 11.5–13 weeks [34,35]. Growth plates of skeletally immature 3- and 10-week-old mice were therefore chosen as examples of sites of active endochondral ossification to study the expression of the BAG-1 protein. Growth plates in 3-week-old mice demonstrated well-defined regions of chondrocyte differentiation namely, reserve (RZ), proliferative (PZ), upper hypertrophic/maturing/prehypertrophic (UHZ) and hypertrophic (HZ) zones (Fig. 2A). At 3 weeks, BAG-1 expression in the growth plate was predominantly observed in the chondrocytes of the reserve and upper hypertrophic zones (Fig. 2A). In comparison, few hypertrophic chondrocytes were found to express the BAG-1 protein, and BAG-1 expression was largely excluded from the chondrocytes of the proliferative zone (Fig. 2A). At 10 weeks, growth plates were significantly shorter and the zones of chondrocyte differentiation became less pronounced in comparison to the growth plates of 3-week-old mice (Fig. 2B). BAG-1 was expressed by majority of the upper hypertrophic/prehypertrophic and hypertrophic chondrocytes, and absent from the proliferative chondrocytes in growth plates of 10-week-old mice (Fig. 2B). Although the 3-week time point utilised in the present study was an earlier time point compared to the 6-week time point used by Kinkel and co-workers [18], findings of this study on the pattern of BAG-1 expression in growth plates of 3-week-old mice reflect those of the previous study in growth plates of 6-week-old mice. The next time point studied by Kinkel and co-workers was 6 months, where the pattern of BAG-1 expression in the growth plate was random [18], unlike the 10-week time point utilised in the present study, where the pattern of BAG-1 expression was found to shift predominantly to the upper hypertrophic and hypertrophic zones of the growth plate. Expression of BAG-1 during the course of ATDC5 cell differentiation in a monolayer culture system Previous studies have demonstrated the efficacy of monolayer cultures of the murine chondrocytic ATDC5 cell line in the presence of insulin to study the molecular mechanisms underlying regulation of chondrocyte differentiation during endochondral bone formation [36–38]. Monolayer cultures of ATDC5 cells grown in the presence of insulin, transferrin and selenium over a period of 28 days (Fig. 3A) were therefore

Fig. 5. Western blot probed with the α-HA antibody demonstrating overexpression of HA–BAG-1L isoform in ATDC5 cells 24 hours post-transfection with 1 μg and 2 μg of mouse pBag-1L expression construct (A). The lanes were marked EV (for cells representing the empty vector control), L (for cells transfected with the pBag-1L expression construct) and with the concentrations, namely 1 μg and 2 μg, at which the expression constructs were used in the transfections. Both BAG-1L and BAG-1S isoforms were expressed from the pBag-1L expression construct. Expression of the BAG-1S protein from the pBag-1L vector, however, was at an appreciably low level in comparison to the BAG-1L protein. Changes in expression of Sox-9, Bcl-2, Col2a1, Runx-2 and Alkaline phosphatase genes were studied in response to BAG-1L overexpression by qPCR (B). Relative expression levels for each gene were normalised to the expression of the β-Actin gene. For each gene, the group with the highest expression was assigned a value of 1 and expression levels in the remaining groups were determined relative to the group exhibiting the highest expression. Fold relative expression levels were expressed as mean ± SD (n = 3) for plotting as bar graphs. Overexpression of the BAG-1L protein did not alter endogenous Sox-9 and Bcl-2 gene expression. Expression of the Col2a1 transcript was down-regulated as a result of BAG-1L overexpression. The BAG-1L overexpressed protein was found to enhance expression of the Runx-2 and Alkaline phosphatase transcripts. ⁎⁎⁎P b 0.001.

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utilised in the present study as an in vitro model of endochondral ossification to monitor the expression of BAG-1 in relation to the chondrogenic differentiation markers. ATDC5 cells were ap-

proximately 60% confluent on day 2 of culture and were found to reach confluence on day 7, which served as a stimulus for further differentiation and progressive hypertrophy. Formation

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of cartilaginous nodules was observed on day 14 of culture and well-defined cartilaginous nodules were detected by day 21 of culture. The cartilaginous nodules were found to grow and increase in size by day 28 of culture. Although mineralization was not examined in day 28 cultures in the present study, the presence of mineralizing matrix vesicles in the extracellular matrix in day 28 monolayer cultures of ATDC5 cells differentiated in the presence of FCS, insulin, transferrin and selenium has been demonstrated in a previous study [37]. By electron microscopy and electron probe microanalysis of ATDC5 cell cultures, this study showed that calcification was first initiated in the matrix vesicles in the territorial matrix and it then advanced progressively along the collagen fibres in a manner similar to that occurring in vivo. Expression of typical chondrocytic proteins (Fig. 3B) and their mRNA transcripts (Fig. 3C) was analysed during the course of ATDC5 cell differentiation by Western blotting and qPCR, respectively. Expression of SOX-9 and Type II collagen proteins and their mRNA transcripts was observed in day 2 cultures, peaked at day 7 and was detected until day 21 of culture. Day 28 mineralised cultures, however, were characterised by significant downregulation of expression of the SOX-9 and Type II collagen proteins and their mRNA transcripts. Expression of the BCL-2 protein and its mRNA transcript, observed initially in day 2 cultures, was found to increase steadily up until day 14 of culture, following which both protein and gene expression decreased in day 21 cultures and was almost absent in day 28 cultures. As expression of Sox-9, Col2a1 and Bcl-2 has been shown to be downregulated at the onset of hypertrophy, day 28 cultures of ATDC5 cells were characterised by the presence of terminally differentiated/hypertrophic chondrocytes. This was confirmed in the present study by way of a steady increase in expression of the Col10a1 transcript during the time course of ATDC5 differentiation, with maximal expression observed in day 28 cultures. Thus, based on morphological examination of phase contrast images of ATDC5 cultures and analysis of expression of typical chondrocyte differentiation markers, cultures up until day 14 represented the proliferative phase of chondrocyte differentiation, day 21 cultures were characterised by chondrocyte maturation and the onset of hypertrophy, and day 28 cultures exhibited terminal differentiation. In comparison to the expression profiles of the different chondrocytic markers, expression of both BAG-1 isoforms

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(50 kDa and 32 kDa) and the Bag-1 mRNA transcript (Figs. 3B and C) was maintained throughout the course of ATDC5 differentiation. Significantly, BAG-1 protein and mRNA expression was observed in hypertrophic chondrocytes of day 28 mineralised cultures in presence of high Col10a1 expression and negligible Sox-9, Col2a1 and Bcl-2 expression. In addition to the bands representing the 50 kDa and 32 kDa BAG-1 isoforms, the rabbit α-rodent BAG-1 antibody used in this study was able to detect another band of ∼ 46 kDa (denoted by a star). The 46 kDa BAG-1M isoform, normally expressed in human cells, has not been reported in murine cells [29]. This additional ∼46 kDa band observed in ATDC5 cells was probably as a result of the antibody cross-reacting with other members of the BAG-family of proteins, described in previous studies [21]. BAG-1 functions as an anti-apoptotic defence against heat-shock in chondrocytes ATDC5 cells overexpressing GFP–BAG-1L and GFP– BAG-1S proteins were subjected to heat-shock at 48 °C for 30 min to determine whether overexpression of the BAG-1 proteins protected chondrocytic cells against apoptosis due to heat-shock. Both BAG-1 isoforms (L and S) were expressed from the mouse pEGFP–Bag-1L expression construct, used to transfect ATDC5 cells, as the initiation codon (ATG) for translation of the BAG-1S isoform was not modified in this construct (S. Wiese, unpublished observations). The efficiency of transfection was approximately 40%. Expression of the GFP– BAG-1L (nuclear localisation) and GFP–BAG-1S (cytosolic and nuclear localisation) proteins was observed in ATDC5 cells (subjected to heat-shock) up until 48 h post-transfection with the pEGFP–Bag-1L (1 μg/well) expression construct (Figs. 4A and C). Parallel image of ATDC5 cells with DAPI-stained nuclei (Fig. 4B). In control ATDC5 cultures transfected with the pEGFP empty vector (1 μg/well), expression of GFP was observed in the cell nuclei (stained with DAPI) and cytoplasm (Figs. 4D–F). This suggested that the GFP protein alone was able to localise at sub-cellular sites within the ATDC5 cell similar to the BAG-1 proteins, and hence mask the localisation of the BAG-1 proteins. Thus, overexpression of the BAG-1 isoforms in ATDC5 cells up until 48 h post-transfection was demonstrated by Western blot analysis, and it was possible to detect higher intensity bands representing BAG-1L and BAG-1S proteins in lysates of ATDC5 cells transfected with 1 μg/well of

Fig. 6. Western blots probed with α-FLAG and α-SOX-9 antibodies demonstrating overexpression of the FLAG–SOX-9 protein in ATDC5 cells 24 h post-transfection with 1 μg and 2 μg of the human pSox-9 expression construct (A). EV represented the empty vector control. Overexpression of SOX-9 at both concentrations resulted in significant upregulation of the Bag-1 transcript (B). Relative expression levels for the Bag-1 gene were normalised to the expression of the β-Actin gene. The group with the highest expression was assigned a value of 1 and expression levels in the remaining groups were determined relative to the group exhibiting the highest expression. Fold relative expression levels were expressed as mean ± SD (n = 3) for plotting as bar graphs. To determine the effect of SOX-9 on the Bag-1 gene promoter, CHO cells were co-transfected with 1 μg/well of the human pBag-1–Luciferase promoter reporter construct and varying concentrations of the human pSox-9 expression vector. Blots probed with α-FLAG and α-SOX-9 antibodies demonstrated expression of the FLAG–SOX-9 protein in CHO cells 24 h post-transfection with 400, 600 and 800 ng/well of the pSox-9 expression construct (C). Expression of the SOX-9 protein was not observed in CHO cells transfected with the empty pcDNA3.1 vector or with 200 ng/well of pSox-9 (C). Readings for Firefly Luciferase were divided by those for Renilla Luciferase for each sample, and mean values for each group were plotted as relative light units ± SD; n = 6. The basal level of Bag-1 promoter activity (represented by the dotted bar in panel D) was unaltered by the empty pcDNA3.1 vector (represented by the bar with thick oblique lines in panel D). Activity of the Bag-1 promoter was significantly enhanced by the SOX-9 protein in CHO cells transfected with 400, 600 and 800 ng/well of the pSox-9 expression construct (represented by bars with thin oblique lines in panel D). In the control experiment, activity of the promoterless pGL3-Basic vector (represented by the first grey bar in panel D) was unaltered by SOX-9 expression as a result of transfections with 400 ng/well pSox-9 (represented by the second black bar in panel D). ⁎P b 0.05, ⁎⁎P b 0.01.

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the pEGFP–Bag-1L expression construct compared to the pEGFP empty vector (EV) control (Fig. 4G). The number of apoptotic cells in each group (3 wells per group) was determined by averaging the cell count for apoptotic cells (stained pink with the APOPercentage dye) in five random fields per group, and the results for each group were plotted in the bar graph as mean ± SD (Fig. 4R). Few apoptotic cells were observed in cultures of ATDC5 cells that were transfected with either the pEGFP empty vector (Figs. 4H and I) or the pEGFP– Bag-1L expression construct (Figs. 4J and K), but were not subjected to heat-shock. The number of apoptotic cells in cultures of ATDC5 cells transfected with the pEGFP empty vector (27 ± 12 apoptotic cells out of a total of 1171 ± 20 cells) was not significantly different from the number of apoptotic cells in cultures of ATDC5 cells transfected with the pEGFP–Bag-1L expression construct (34 ± 10 apoptotic cells out of a total of 1162 ± 21 cells). The extent of apoptosis as a consequence of heat-shock in ATDC5 cells transfected with either the pEGFP empty vector (Figs. 4L–N) or the pEGFP–Bag-1L expression construct (Figs. 4O–Q) was significantly higher (⁎⁎⁎P b 0.001) in comparison to the control groups of pEGFP/pEGFP–Bag-1Ltransfected ATDC5 cells not subjected to heat-shock (Figs. 4H and I, and Figs. 4J and K, respectively). The level of heat-shockinduced apoptosis in ATDC5 cells overexpressing BAG-1 (563 ± 36 apoptotic cells out of 1156 ± 26 cells, Figs. 4O–Q), however, was significantly lower (⁎⁎⁎P b 0.001) in comparison to the level of apoptosis as a result of heat-shock in ATDC5 cells transfected with the pEGFP empty vector (1147 ±37 apoptotic cells out of 1159 ± 34 cells, Figs. 4L–N). Effect of BAG-1L overexpression on transcription of genes important in chondrocyte differentiation In order to determine the effect of overexpression of the BAG-1L isoform on the expression of genes important in chondrocyte differentiation, ATDC5 cells were transfected with the mouse pBag-1L (HA-tagged) expression construct at concentrations of 1 μg and 2 μg/100 mm plate. Overexpression of the BAG-1L protein in ATDC5 cells, 24 h post-transfection, was confirmed by Western blot analysis using the α-HA antibody. As illustrated by the intensity of the bands, level of the HA-BAG-1L protein in cells transfected with 2 μg of pBag1L expression construct was higher than in cells transfected with 1 μg of pBag-1L expression construct (Fig. 5A). As the initiation codon (ATG) for translation of the BAG-1S isoform in the pBag1L expression construct was not modified, both BAG-1L and BAG-1S isoforms were expressed from the pBag-1L expression construct (S. Wiese, unpublished observations). Expression of the BAG-1S protein from the pBag-1L vector however, was at a significantly low level in comparison to the expression of the BAG-1L protein as illustrated by the intensity of the bands in the lanes marked L 2 μg and L 1 μg (Fig. 5A). Changes in expression of Sox-9, Bcl-2, Col2a1, Runx-2 and Alkaline phosphatase genes were studied in response to BAG1L overexpression (Fig. 5B). Expression levels of the genes in untransfected cells (Ctrl.) matched those in cells transfected with the empty pcDNA3.1 vector (EV), indicating that the

empty pcDNA3.1 vector had no role to play in altering expression of the studied genes. Overexpression of the BAG-1L protein did not alter endogenous Sox-9 and Bcl-2 gene expression. Expression of the Col2a1 transcript was downregulated as a result of BAG-1L overexpression (⁎⁎⁎P b 0.001). The BAG-1L-overexpressed protein was found to enhance expression of the Runx-2 (⁎⁎⁎P b 0.001) and Alkaline phosphatase (⁎⁎⁎P b 0.001) transcripts. Effect of SOX-9 on Bag-1 gene transcription To elucidate whether SOX-9 influenced expression of the Bag-1 gene, ATDC5 cells were transfected with the human pSox-9 (FLAG-tagged) expression construct at concentrations of 1 μg and 2 μg/100 mm plate. As the mouse (NM_011448) and human (NM_000346) SOX-9 proteins exhibit a homology of approximately 97%, the human SOX-9 protein was overexpressed in murine ATDC5 cells. Overexpression of SOX-9 in ATDC5 cells, 24 h post-transfection, was confirmed by Western blot analysis. When the blot was probed with the α-FLAG antibody, expression of the FLAG–SOX-9 protein was observed in cells transfected with the pSox-9 (FLAG-tagged) expression construct at both concentrations i.e. 1 μg and 2 μg (Fig. 6A). When the blot was probed with the α-SOX-9 antibody, the empty vector (EV) control served to establish basal levels of endogenous SOX-9 protein expression in the ATDC5 cells. It was also possible to demonstrate overexpression of the SOX-9 protein using the α-SOX-9 antibody as the intensity of bands for SOX-9 in cells transfected with the pSox-9 expression construct was higher in comparison to the intensity of the band representing the endogenous SOX-9 protein in the EV control (Fig. 6A). A control (Ctrl.) sample comprising of untransfected cells was used to determine baseline endogenous Bag-1 gene expression. Expression levels for Bag-1 in untransfected cells (Ctrl.) matched those in cells transfected with the empty pcDNA3.1 vector (EV), indicating that the empty pcDNA3.1 vector had no role to play in altering expression of the Bag-1 gene (Fig. 6B). Transcription of the Bag-1 gene was significantly increased (⁎P b 0.05) as a result of overexpression of SOX-9 (Fig. 6B). To confirm the above observation and to determine whether SOX-9 regulated activity of the Bag-1 gene promoter, CHO cells were co-transfected with 1 μg/well of the human pBag-1– Luciferase promoter reporter construct and varying concentrations of the human pSox-9 (FLAG-tagged) expression vector. Blots probed with α-FLAG and α-SOX-9 antibodies demonstrated expression of the FLAG–SOX-9 protein in CHO cells 24 h post-transfection with 400, 600 and 800 ng/well of the pSox-9 expression construct (Fig. 6C). Expression of the SOX-9 protein was not observed in CHO cells transfected with 200 ng/well of pSox-9 (Fig. 6C). CHO cells were not found to express any endogenous SOX-9 protein as indicated by the lack of expression in the EV lane (Fig. 6C). Bag-1 promoter activity was determined by measuring the activity of the Luciferase reporter gene. The basal level of Bag-1 promoter activity (represented by the dotted bar) was unaltered by the empty pcDNA3.1 vector (Fig. 6D). A significant increase (⁎P b 0.05, ⁎⁎P b 0.01) in the activity of the

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Bag-1 promoter, stimulated by the SOX-9 protein, was observed in CHO cells transfected with 400, 600 and 800 ng/well of the pSox-9 expression construct (Fig. 6D). In the control experiment, activity of the promoterless pGL3-Basic vector, used to clone the human Bag-1 promoter, was unaltered by SOX-9 expression as a result of transfections with 400 ng/well pSox-9 (Fig. 6D). Discussion In the present study, expression of BAG-1 in long bones of skeletally immature mice was reported in cells of both the chondrogenic as well as the osteogenic lineages. Expression of both BAG-1L and BAG-1S isoforms was demonstrated during early stages of osteogenic differentiation, marked by the presence of Alkaline phosphatase, in day 12 cultures of mouse bone marrow-derived cells. BAG-1 proteins were expressed in fully differentiated osteoblasts of the primary spongiosa and expression maintained even in osteocytes within the bone shaft. It was therefore possible to show expression of BAG-1 in osteogenic cells at various stages of differentiation. The BAG-1 protein was immunolocalised to the perichondrium, a region characterised by the presence of progenitor cells of both the osteogenic as well as the chondrogenic lineages [39]. Although expression of BAG-1 was previously reported in articular and growth plate cartilage in long bones of mice [18], this study demonstrated localisation of the BAG-1 protein at additional sites within mouse long bones not previously reported. We have demonstrated in the present study that overexpression of BAG-1L and -S isoforms protected ATDC5 cells against heat-shock-induced apoptosis. BAG-1 has been implicated in a number of important cellular control pathways that protect cells from a range of apoptotic stimuli [40–42]. Previous studies have elucidated the anti-apoptotic function of BAG-1 in a number of cell types namely, photoreceptors [43], cardiac myocytes [44], neurons and haematopoietic cells [31,45]. Stress conditions like heat-shock result in the production of heat-shock proteins, which assist cells in combating stress-induced damage [46]. The BAG domain of BAG-1 mediates interaction with the HSP70-family molecular chaperones and RAF-1 kinase [47,48], while the ubiquitin-like domain (ULD) is essential for interaction with the proteosome, thereby facilitating the BAG-1 protein to link the heat-shock proteins to the proteosome for protein degradation [41]. The BAG-1S isoform (both endogenous and overexpressed) has been reported to relocate from the cytoplasm to the nucleus as a result of heat-shock, and both the BAG-1S isoform and the constitutively-nuclear localised BAG-1L isoform suppressed heat-shock-induced apoptosis to the same extent [27,44]. As chondrocytes are isolated within lacunae in vivo, they resort to modes of cell death morphologically distinct from classical apoptosis [2–8]. On rare occasions however, the hallmarks of classical apoptosis have been observed in hypertrophic growth plate chondrocytes in vivo [3]. A significant number of chondrocytes with characteristic features of classical apoptosis have been observed in in vitro monolayer cultures [49,50], possibly because chondrocytes are not isolated within lacunae in this culture system. In the present study, ATDC5 cells served as an in vitro model to demonstrate the anti-apoptotic role

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of BAG-1 in chondrocytic cells subjected to heat-shock. Heatshock has been shown to induce morphological changes consistent with classical apoptosis in rat articular chondrocytes [51]. ATDC5 cells subjected to heat-shock were not examined in the present study for the alternative modes of cell death opted by terminally differentiated chondrocytes in vivo. For the results to be relevant within the context of an in vivo scenario, as a part of the future work, we will have to overexpress the BAG-1 protein in chondrocytic cells representing a terminally differentiated phenotype and also confirm that the modes of cell death in this in vitro system are comparable to the in vivo scenario. Results of the present study on the anti-apoptotic role of BAG-1 in ATDC5 cells however, serve as a starting point to enhance our understanding on the possible roles of this protein in chondrocytes. It has been suggested that BAG-1 proteins affect transcription of genes either positively or negatively depending upon cell type and context [52]. The N-terminal decapeptide characterised by two clusters of three basic amino acids each, has been shown to facilitate association of the BAG-1L and BAG-1M proteins with DNA in vitro [53]. Overexpression of HAP50 (the large/50 kDa BAG-1 isoform in humans), in various cell types by transient transfection, enhanced transcription of reporter genes coupled to different promoters [54]. Overexpression of HAP50 in cells under non-stress conditions was found to increase expression of endogenous genes coding for heat-shock proteins and transcription factors like c-Jun and c-Fos, essentially proteins vital for cell viability, differentiation and apoptosis [54]. BAG-1S was demonstrated to be incapable of functioning as a transcriptional regulator. Although addition of an exogenous nuclear-targeting sequence to BAG-1S was sufficient to force it into the nucleus, the protein failed to function as a transcriptional regulator [55]. Thus, the BAG-1L isoform was overexpressed in ATDC5 cells to determine whether it had a role in regulating the transcription of genes important in chondrocyte differentiation and endochondral ossification. BAG-1L suppressed Col2a1 gene expression and enhanced transcription of Runx-2 and Alkaline phosphatase genes. Induction of Col2a1 expression occurs with the change in cellular phenotype from prechondrogenic cells to proliferating chondrocytes [11], while expression of Col2a1 declines in hypertrophic chondrocytes [12]. Although Runx-2 [56,57] and Alkaline phosphatase [58,59] are acknowledged as early osteogenic markers, Runx-2 is also expressed by hypertrophic chondrocytes and it functions as a positive regulatory factor in chondrocyte maturation [38], while Alkaline phosphatase expression by hypertrophic chondrocytes is implicated in the mineralization process [60]. Thus, overexpression of BAG-1L in chondrocytic cells resulted in an increase in transcription of genes normally expressed at the onset of hypertrophy and cartilage mineralization in the process of endochondral ossification. In our studies with ATDC5 monolayer cultures, noticeable expression of the Col10a1 transcript was first detected at day 14 i.e. 7 days after the cells reached confluence. Previous work by Enomoto and co-workers with ATDC5 monolayer cultures has also demonstrated initiation of Col10a1 expression 9 days after confluence [38]. It was therefore not possible to determine the effect of BAG-1L overexpression on Col10a1 gene transcription in our transient

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transfection experiments, where ATDC5 cells were transfected at 40% confluency. HAP50 (the 50 kDa BAG-1 isoform in humans) has been shown to enhance the transcription of heat-shock proteins under non stress conditions in a number of cell types [54]. Heat-shock proteins (HSP28, HSP70 and HSP110) have been demonstrated to be expressed under non-stress conditions in chondrocytes of the reserve, upper hypertrophic and hypertrophic zones of rodent growth plates [61,62]. Expression of heat-shock proteins by growth plate chondrocytes has been related to the terminal differentiation of these cells, as heat-shock proteins promote hypertrophy and calcification by stopping protein synthesis in chondrocytes that possess terminal differentiation potency [61]. Expression of BAG-1 in chondrocytes of reserve, upper hypertrophic and hypertrophic zones of the growth plates of 3- and 10-week-old mice mimicked heat-shock protein expression in rodent growth plates. We also demonstrated that overexpression of BAG-1L in chondrocytic cells downregulated expression of Col2a1, a gene expressed by prehypertrophic chondrocytes, and promoted expression of Runx-2 and Alkaline phosphatase, genes associated with chondrocyte hypertrophy and cartilage mineralization/calcification. Based on these observations, expression of BAG-1 and heat-shock proteins by growth plate chondrocytes assists in chondrocyte hypertrophy and cartilage mineralization, thereby facilitating the process of endochondral ossification. The present study also demonstrated regulation of Bag-1 gene expression in chondrocytes. The SOX-9 protein was found to enhance activity of the Bag-1 gene promoter. The optimal SOX-9 DNA-binding sequence, AGAACAATGG, comprises of a core/ consensus DNA-binding element AACAAT flanked by 5′ AG and 3′ GG nucleotides [63]. Although not a perfect match with the SOX-9 consensus DNA-binding element, we were able to identify sequences, AAGAAT (nucleotides −556 to −551) and AACAAG (nucleotides −13 to −8), in the human Bag-1 gene promoter to which the SOX-9 protein could possibly bind. Future work involving electrophoretic mobility shift assays and ChIP assays will offer insight into this interaction between the SOX-9 protein and the Bag-1 gene promoter. As the effect of SOX-9 on Bag-1 gene transcription was modest, the likelihood of other factors contributing to the expression of Bag-1 in chondrocytes cannot be overruled. TGF-β may represent one such factor considering the Bag-1 gene promoter is characterised by a TGF-β response element [33]. Future work will attempt to explore this aspect of Bag-1 gene regulation. SOX-9 has been identified as a potent activator of the chondrocyte-specific enhancer associated with the Col2a1 gene [10]. In light of our observation that BAG-1 downregulated Col2a1 expression, it was surprising to observe an increase in Bag-1 expression as a result of SOX-9 overexpression. To explain these contradictory results, we would like to suggest that robust expression of SOX-9 [13] and BCL-2 [15] in proliferating chondrocytes is sufficient to maintain Col2a1 expression [15,64] in these cells in presence of low BAG-1 expression. The onset of hypertrophy is characterised by high BAG-1 protein expression and downregulation of Sox-9 [13] and Bcl-2 [15] expression. In this scenario, BAG-1 assists the onset of hypertrophic differentiation and subsequent cartilage mineralization by suppressing

expression of Col2a1 and inducing expression of Runx-2 and Alkaline phosphatase. In conclusion, a novel finding of this study is the role of BAG1 as a transcriptional regulator of genes involved in chondrocyte hypertrophy and cartilage mineralization during the process of endochondral ossification. Additionally, we have demonstrated for the first time the regulation of Bag-1 gene expression by SOX9 and the anti-apoptotic role of BAG-1 in chondrocytes. The present study therefore provides a starting point to enhance our understanding on the role of BAG-1 in endochondral ossification. Modulation of Bag-1 expression can mediate chondrocyte differentiation and turnover, and offer further insight into the molecular regulation of endochondral ossification. Acknowledgments The authors would like to acknowledge Dr Stefan Wiese for providing the pBag-1 expression constructs, Dr Tim Hardingham for the pSox-9 expression construct and, Dr Xialong Yang and Dr Shou-Ching Tang for providing the Bag-1 promoter construct. The authors would also like to thank Dr Agi Grigoriadis for the generous donation of ATDC5 cells. The excellent technical expertise of Mrs Michelle Bryson, the assistance provided by Dr Bram Sengers in image analysis and useful discussions with Dr Trudy Roach are acknowledged by the authors. Finally, the authors would like to thank the Biotechnology and Biological Sciences Research Council (BBSRC) for fellowship support to R. Tare and research support to R. Oreffo and P. Townsend. References [1] Poole AR. The growth plate: cellular physiology, cartilage assembly and mineralisation. In: Hall BK, Newman SA, editors. Cartilage: molecular aspects. Boca Raton, Florida: CRC Press; 1991. p. 179–211. [2] Schweichel JU, Merker HJ. The morphology of various types of cell death in prenatal tissues. Teratology 1973;7:253–66. [3] Roach HI, Clarke NM. Physiological cell death of chondrocytes in vivo is not confined to apoptosis. New observations on the mammalian growth plate. J Bone Joint Surg Br 2000;82:601–13. [4] Wilsman NJ, Farnum CE, Hilley HD, Carlson CS. Ultrastructural evidence of a functional heterogeneity among physeal chondrocytes in growing swine. Am J Vet Res 1981;42:1547–53. [5] Carlson CS, Hilley HD, Henrikson CK. Ultrastructure of normal epiphyseal cartilage of the articular–epiphyseal cartilage complex in growing swine. Am J Vet Res 1985;46:306–13. [6] Ahmed YA, Tatarczuch L, Pagel CN, Davies HM, Mirams M, Mackie EJ. Physiological death of hypertrophic chondrocytes. Osteoarthr Cartil 2007;15:575–86. [7] Roach HI, Aigner T, Kouri JB. Chondroptosis: a variant of apoptotic cell death in chondrocytes? Apoptosis 2004;9:265–77. [8] Shapiro IM, Adams CS, Freeman T, Srinivas V. Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate. Birth Defects Res C Embryo Today 2005;75:330–9. [9] Wright E, Hargrave MR, Christiansen J, Cooper L, Kun J, Evans T, et al. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet 1995;9:15–20. [10] Lefebvre V, Huang W, Harley VR, Goodfellow PN, de Crombrugghe B. SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene. Mol Cell Biol 1997;17:2336–46.

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