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Originally published In Press as doi:10.1074/jbc.M605937200 on August 16, 2006

J. Biol. Chem., Vol. 281, Issue 41, 30848-30856, October 13, 2006
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Annexin V/beta5 Integrin Interactions Regulate Apoptosis of Growth Plate Chondrocytes*

Wei Wang and Thorsten Kirsch1

From the Musculoskeletal Research Laboratories, Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received for publication, June 21, 2006 , and in revised form, August 9, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C {alpha} (PKC{alpha}), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKC{alpha} play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKC{alpha} was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKC{alpha} led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKC{alpha} stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
During endochondral ossification, the bone structures are first cartilaginous. Chondrocytes in these growth plate cartilages undergo a series of differentiation events, including proliferation, hypertrophy, and terminal differentiation, eventually leading to the replacement of mineralized cartilage by bone. Evolving in vitro and in vivo evidence shows that the final fate of terminally differentiated growth plate chondrocytes is apoptosis (programmed cell death) (1, 2). We have demonstrated that retinoic acid treatment of growth plate chondrocytes stimulates terminal differentiation events and eventually leads to apoptosis of these cells (3, 4). In addition, several in vivo studies have also revealed that apoptosis is the final fate of terminally differentiated growth plate chondrocytes. For example, we and others have demonstrated that apoptosis of growth plate chondrocytes occurs in chondrocytes at the chondro-osseous junction in chicken growth plate and sternal cartilage (1, 5). Furthermore, mice with targeted disruptions of both alleles for the antiapoptotic protein bcl-2 have short limbs and accelerated ossification of their growth plates (6). In addition, two chondrodysplastic conditions (parathyroid hormone-related peptide knock-out mice and activating mutations of the fibroblast growth factor receptor-3 (FGFR-3)) are associated with increased apoptosis of the growth plate chondrocytes (7, 8).

The various differentiation events of growth plate chondrocytes, including apoptosis, are precisely regulated to allow coordinated longitudinal bone growth. A disturbance in the regulation of these events leads to growth retardation. For example, glucocorticoid treatment results in growth retardation by decreasing the proliferation rate of growth plate chondrocytes and by increasing the apoptosis rate of terminally differentiated growth plate chondrocytes (9, 10). Therefore, the understanding of the mechanisms regulating the various differentiation events is highly relevant. However, very little is known about the regulation of apoptosis of growth plate chondrocytes. Recent studies have shown that annexin V, a cytosolic protein that binds to membranes in the presence of calcium, binds to the cytoplasmic domain of beta5 integrin and to active protein kinase C {alpha} (PKC{alpha})2 and that these interactions play a key role in the regulation of apoptosis of endothelial cells (11, 12). Interestingly, annexin V and beta5 integrin are expressed in hypertrophic and terminally differentiated growth plate chondrocytes (13, 14). Therefore, we hypothesized that the interactions among annexin V, beta5 integrin, and PKC{alpha} play a role in the regulation of apoptosis of growth plate chondrocytes. To address this hypothesis, we used a peptide mimic of annexin V, which has been shown to bind to beta5 integrin and induce apoptosis in endothelial cells (11), overexpression of annexin V using a retroviral expression vector, and suppression of annexin V using small interfering RNA (siRNA), and determined cell viability, bcl-2 and bax expression, and caspase-3 activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—The preparation and specificity of antibodies specific for annexin V were described previously (13). Antibodies specific for beta5 integrin subunit were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Penetratin (Pen) peptides (Pen-SDNRYIGSW and Pen-VVISYSMPD) were purchased from Global Peptide Services (Fort Collins, CO). Calphostin C, myristoylated (Myr)-PKC{alpha}/beta, and phorbol 12-myristate 13-acetate (PMA) were purchased from EMD Biosciences/Calbiochem. Myr-PKC{zeta} was purchased from BIO-SOURCE (Camarillo, CA).

Chondrocyte Culture—Chondrocytes were isolated from the hypertrophic zone of day 19 embryonic chick tibia growth plate cartilage as described previously (15). Cells were grown in monolayer cultures in Dulbecco's modified Eagle's medium (Invitrogen) containing 5% fetal calf serum (HyClone, Logan, UT), 2 mM l-glutamine (Invitrogen), and 50 units/ml penicillin and streptomycin (Invitrogen) (complete medium). After 3 days, cells were incubated with high titer retroviral stocks of replication-competent, non-transforming Rous sarcoma virus-based expression vector (RCAS-BP) or RCAS-BP containing full-length annexin V cDNA in a small volume (5 x 106 colonyforming units/106 cells in less than 1 ml of medium) for 4 h. Thereafter, cells were cultured in complete medium until ~90% of chondrocytes were infected. The degree of overexpression was detected by immunoblotting using antibodies specific for annexin V (16). For siRNA experiments, growth plate chondrocytes were transfected with 200 nM siRNA specific for annexin V using Lipofectamine 2000 transfection reagent according to the manufacturer's protocol (Invitrogen) (16). After transfection, cells were cultured in complete medium in the absence or presence of peptides and/or calphostin C (10 nM), PMA (3 µM), Myr-PKC{alpha}/beta (100 µM), or Myr-PKC{zeta} (100 µM).

Construction and Production of Chicken Retrovirus RCAS-BP—Full-length annexin V cDNA was first cloned into an adaptor vector SLAX-myc, which contained a 10-amino acid epitope of human c-Myc tag fused to the carboxyl-terminal end of the recombinant protein and then subcloned into RCAS-BP (17). To obtain viral stocks, the plasmid constructs and RCAS-BP containing no insert were used to transfect chicken embryonic dorsal fibroblasts using the Lipofectamine 2000 transfection reagent as described previously (16).

Construction of siRNA to Silence Annexin V Expression in Growth Plate Chondrocytes—We used the Silencer siRNA construction kit from Ambion, Inc. (Austin, TX) to synthesize siRNA. Four pairs of oligonucleotides encoding the desired sense and antisense siRNA strands were designed according to the chicken annexin V sequence by using a computer program (Ambion Inc.) Oligonucleotides were designed to include an 8-base sequence complementary to the 5' end of T7 promoter primer included in the kit. The procedure was performed as described previously (16). The different siRNAs were tested for the efficiency to suppress annexin V protein expression in 10-day embryonic chicken dorsal fibroblasts to select the most effective siRNAs for transfection of growth plate chondrocytes. The sequences of the most efficient oligonucleotides were: antisense, 5'-AAGCATGCAATCAAGGGAGCACCTGTCTC-3', and sense, 5'-AATGCTCCCTTGATTGCATGCCCTGTCTC-3'.

Double Immunofluorescence and Peptide Internalization and Visualization—Cells were incubated with 10 µg/ml biotinylated peptides. After 2 h, the cultures were washed with phosphate-buffered saline, fixed, permeabilized with ethanol/acetic acid (9:1, v/v) for 5 min at -20 °C, blocked for 30 min with goat serum, and incubated with fluorescein isothiocyanate-conjugated streptavidin for 1 h. After washing, cells were analyzed by fluorescence microscopy (Nikon). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). For double immunostaining for beta5 integrin and annexin V, cells were washed, fixed with ethanol, and incubated with primary mouse monoclonal antibodies specific for beta5 integrin and rabbit polyclonal antibodies specific for annexin V. After washing, cells were incubated with Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Invitrogen/Molecular Probes) and analyzed by inverted fluorescence microscopy (Nikon). For double immunostaining of 10-µm-thick paraffin sections of day 19 embryonic chicken growth plate, sections were deparaffinized, rehydrated, and then pretreated with 2 mg/ml sheep testicular hyaluronidase (Sigma) in phosphate-buffered saline for 30 min at 37 °C. After blocking, sections were immunostained with primary and secondary antibodies as described above.

Caspase-3 Activity and Cell Viability Assays—Caspase-3 activity was measured using the ApoAlert caspase fluorescent assay kit (Clontech) as described previously (4). Caspase-3 activity was normalized to the protein content in each culture. Caspase-3 activity is expressed as relative units, with caspase-3 activity of the untreated or uninfected culture set as 1. Cell viability was determined by measurement of the cellular metabolism of MTT (Sigma) following the manufacturer's protocol.

SDS-PAGE and Immunoblotting—To determine the amount of annexin V in growth plate chondrocytes, cells were lysed in NuPAGE SDS sample buffer (Invitrogen). Thirty micrograms of total protein was subjected to SDS-PAGE and immunoblotting. Before electrophoresis, the reducing agent was added to the sample solution, denatured at 70 °C for 10 min, and analyzed by electrophoresis in 10% Bis-Tris gels following the NuPAGE electrophoresis protocols. Samples were electro-blotted onto nitrocellulose filters after electrophoresis. After blocking with a solution of low fat milk protein, blotted proteins were immunostained with primary antibodies and then peroxidase-conjugated secondary antibodies, and the signal was detected by enhanced chemiluminescence (Pierce).

Statistical Analysis—Numerical data are presented as mean ± S.D. (n ≥ 3), and statistical significance between groups was identified using the two-tailed Student's t test (p values are reported in the figure legends).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The beta5 integrin subunit was expressed in hypertrophic growth plate cartilage (Fig. 1A). Double immunostaining revealed that cells in type X collagen-positive regions were immunostained with antibodies specific for the beta5 integrin subunit (Fig. 1A), whereas the proliferative zone of growth plate cartilage showed no staining for type X collagen and beta5 integrin (Fig. 1A). Type X collagen staining was localized in the extracellular matrix, whereas beta5 integrin staining was cellular. Annexin V is also expressed in the hypertrophic region of growth plate cartilage (Fig. 1A) (see also Ref. 13). Thus, annexin V, beta5 integrin, and type X collagen are highly expressed in the hypertrophic region of growth plate cartilage. A previous study has shown that annexin V binds to the cytosolic domain of the beta5 integrin subunit (11). Double immunostaining with antibodies specific for beta5 integrin and annexin V revealed a co-localization of both proteins in hypertrophic growth plate chondrocytes in culture (Fig. 1B), confirming previous findings showing an interaction between annexin V and the beta5 integrin subunit (11).


Figure 1
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  		FIGURE 1.
Immunostaining of sections from day 19 embryonic chicken growth plate cartilage with antibodies specific for type X collagen, beta5 integrin, and annexin V and growth plate chondrocytes in culture with antibodies specific for annexin V (AnV) and beta5 (beta5) integrin. A, sections of day 19 embryonic chicken growth plate cartilage were double-stained with mouse anti-beta5 integrin and rabbit anti-type X collagen immunoglobulin G and then by Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies. Sections were also stained with antibodies specific for annexin V. Note that positive immunostaining for beta5 integrin (green) and type X collagen (red) was co-detected in the hypertrophic (Hyp.) but not in the proliferative (Prolif.) zone of growth plate cartilage. Annexin V was also detected in the hypertrophic zone but not in the proliferative zone. B, growth plate chondrocytes isolated from the hypertrophic zone of day 19 embryonic chicken growth plate cartilage were double-stained with rabbit anti-annexin V and mouse anti-beta5 integrin and then by Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies. Note the co-localization of annexin V (green) and beta5 integrin (red) in growth plate chondrocytes (Merged). Bar, 100 µm.

 
To determine the role of annexin V and its interactions with beta5 integrin, we overexpressed annexin V in hypertrophic growth plate chondrocytes using the retroviral expression vector RCAS-BP. Infection of growth plate chondrocytes with RCAS-BP containing full-length annexin V cDNA (Fig. 2A, AnV/RCAS) resulted in a notable increase (2-3-fold) of annexin V protein expression when compared with the expression levels of uninfected or RCAS-BP-infected (Fig. 2A, Uninfected, RCAS) growth plate chondrocytes. Overexpression of full-length annexin V led to a decrease of bcl-2 expression and increases of bax expression and caspase-3 activity when compared with the levels of RCAS-infected cells (Fig. 3, A and B, AnV/RCAS, RCAS). To determine whether annexin V and beta5 integrin interactions are involved in mediating apoptotic events in growth plate chondrocytes, we used a peptide mimic of annexin V (VVISYSMPD) that has been shown to bind to beta5 integrin and trigger cell death of endothelial cells (11). We synthesized an internalizing version of the peptide by using the Pen system for intracellular delivery. Pen, a peptide containing 16 amino acids that are part of the third helix of the Antennapedia protein homeodomain, has translocating properties and therefore is capable of carrying hydrophilic compounds across the plasma membrane and delivering them to the cytoplasmic space without degradation (18). We fused the peptide mimic of annexin V (VVISYSMPD) or a control unrelated peptide (SDNRYIGSW) to Pen and added a biotin moiety to visualize internalization. Both Pen-VVISYSMPD peptide and the control peptide were internalized and uniformly distributed in the cytoplasm (Fig. 2B, Pen-SDNRYIGSW, Pen-VVISYSMPD). Cells not incubated with these peptides showed no cytoplasmic fluorescence staining (Fig. 2B, Control). Treatment of growth plate chondrocytes with 12 µM Pen-VVISYSMPD for 6 h resulted in a decreased expression of the antiapoptotic factor bcl-2 and an increased expression of the apoptotic factor bax when compared with the expression levels of untreated cells (Fig. 3A, Pen-VVISYSMPD, Control). Cells treated with the control peptide showed similar expression levels of bcl-2 and bax as untreated cells (Fig. 3A, Pen-SDNRYIGSW, Control). Treatment of growth plate chondrocytes with 12 µM Pen-VVISYSMPD for 24 h led to a notable increase of caspase-3 activity when compared with untreated cells or cells treated with Pen-SDNRYIGSW (Fig. 3B, Pen-VVISYSMPD, Pen-SDNRYIGSW, Control).

Next we determined whether Pen-VVISYSMPD and beta5 integrin are sufficient to induce apoptotic events in growth plate chondrocytes or whether full-length annexin V is also required. Therefore, we suppressed annexin V expression in Pen-VVISYSMPD-treated growth plate chondrocytes using siRNA technology and measured cell viability and caspase-3 activity. Using annexin V-specific siRNA, annexin V expression was notably suppressed in hypertrophic growth plate chondrocytes (annexin V expression was suppressed by ~70%; Fig. 4, siAnV). Pen-VVISYSMPD treatment reduced the cell viability to ~40% (Fig. 5A, Pen-VVISYSMPD). The control peptide Pen-SDNRYIGSW did not affect cell viability (Fig. 5A, Pen-SDNRYIGSW). Transfecting cells with siRNA specific for annexin V or treatment with Pen-SDNRYIGSW (control peptide) and transfection with annexin V-specific siRNA also did not affect cell viability (Fig. 5A, siAnV, Pen-SDNRYIGSW/siAnV). However, suppression of annexin V expression led to an increase of cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes (Fig. 5A, Pen-VVISYSMPD/siAnV).


Figure 2
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  		FIGURE 2.
Overexpression of AnV using a retroviral expression vector (RCAS) and internalization of Pen-SDNRYIGSW and Pen-VVISYSMPD peptides in growth plate chondrocytes. A, total cell extracts (30 µg of total protein) from uninfected, RCAS-infected (RCAS), and RCAS containing full-length annexin V cDNA-infected (AnV/RCAS) growth plate chondrocytes were subjected to SDS-PAGE and immunoblotting with antibodies specific for annexin V after a 3-day infection. Note the increase in annexin V protein expression in AnV/RCAS-infected cells. beta-actin was used as a control to show equal loading. B, growth plate chondrocytes were treated with the internalizing version of the SDNRYIGSW and VVISYSMPD peptides. Internalization of biotin-labeled and Pen-conjugated annexin V-mimicking peptide (Pen-VVISYSMPD) and an unrelated control (Pen-SDNRYIGSW) into growth plate chondrocytes was visualized using immunostaining with an antibody specific for biotin fused to the amino terminus of Pen after a 4-h treatment. Cell nuclei were counterstained with DAPI. Control consisted of cells not treated with peptides and immunostained with antibody specific for biotin and counterstained with DAPI. Bar, 100 µm.

 


Figure 3
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  		FIGURE 3.
Bcl-2 and bax gene expression and caspase-3 activity of growth plate chondrocytes. A, gene expression of bcl-2 and bax was detected using quantitative real-time PCR in Pen-VVISYSMPD-treated, Pen-SDNRYIGSW-treated, or AnV/RCAS-infected growth plate chondrocytes after a 6-h treatment or a 5-day infection. B, apoptosis was determined by measuring caspase-3 activity after a 24-h treatment with the peptides or a 7-day infection. Treatment with annexin V-mimicking peptide (Pen-VVISYSMPD) or infection with AnV/RCAS resulted in up-regulation of gene expression of bax and caspase-3 activity and down-regulation of bcl-2 gene expression when compared with the levels in untreated (Control), Pen-SDNRYIGSW-treated, or RCAS-infected (RCAS) cells. Data were obtained from triplicated PCR reaction of three different cultures or from caspase-3 activities from three different cultures and were expressed as mean ± S.D. (AnV/RCAS versus RCAS; Pen-VVISYSMPD versus control; *, p ≤ 0.01).

 
Pen-VVISYSMPD treatment resulted in ~5-fold increase of caspase-3 activity in growth plate chondrocytes when compared with the activity of untreated cells (Fig. 5B, Pen-VVISYSMPD, Control). Caspase-3 activity was markedly decreased in Pen-VVISYSMPD-treated cells transfected with annexin V-specific siRNA when compared with the levels in Pen-VVISYSMPD-treated cells (Fig. 5B, Pen-VVISYSMPD/siAnV, Pen-VVISYSMPD). Transfection of growth plate chondrocytes with annexin V-specific siRNA, treatment with Pen-SDNRYIGSW, or treatment with Pen-SDNRYIGSW (control peptide) and transfection with annexin V-specific siRNA had no effect on caspase-3 activity (Fig. 5B, siAnV, Pen-SDNRYIGSW, Pen-SDNRYIGSW/siAnV).

Annexin V has been shown not only to bind to the cytoplasmic domain of the beta5 integrin subunit but also to bind to active PKC{alpha} (11, 12). Binding of annexin V to active PKC{alpha} inhibits PKC{alpha} activity (12, 19). Since active PKC{alpha} has been shown to be involved in mediating cell survival of chondrocytes (20), we asked whether inhibition of PKC{alpha} through annexin V and/or annexin V/beta5 integrin interactions may play a role in mediating apoptosis of growth plate chondrocytes using an activator of PKC (PMA) or generic inhibitors of PKC (calphostin) or subtype-specific PKC inhibitors (Myr-PKC{alpha}/beta, Myr-PKC{zeta}). Treatment of growth plate chondrocytes with PMA, calphostin, or Pen-SDNRYIGSW did not alter cell viability or caspase-3 activity when compared with cell viability and caspase-3 activity of untreated cells (Fig. 6, A and B, PMA, Calphostin, Pen-SDNRYIGSW, Control). Co-treatment with Pen-SDNRYIGSW and PMA or calphostin also did not alter cell viability or caspase-3 activity (Fig. 6, A and B, Pen-SDNRYIGSW/PMA, Pen-SDNRYIGSW/Cal.). PMA further reduced cell viability of Pen-VVISYSMPD-treated cells (from ~40% for Pen-VVISYSMPD-treated cells to ~25% for Pen-VVISYSMPD- and PMA-treated cells) (Fig. 6A, Pen-VVISYSMPD, Pen-VVISYSMPD/PMA), whereas calphostin increased cell viability of Pen-VVISYSMPD-treated cells (~80% viability in Pen-VVISYSMPD and calphostin-treated cells when compared with ~40% viability in Pen-VVISYSMPD-treated cells) (Fig. 6A, Pen-VVISYSMPD, Pen-VVISYSMPD/Cal.). Similar results were obtained for caspase-3 activity. Pen-VVISYSMPD and PMA treatment (Fig. 6B, Pen-VVISYSMPD/PMA) further increased caspase-3 activity when compared with the activity in Pen-VVISYSMPD-treated cells. On the other hand, co-treatment with Pen-VVISYSMPD and calphostin (Fig. 6B, Pen-VVISYSMPD/Cal.) decreased caspase-3 activity to levels similar to those of untreated (Fig. 6B, Control) cells.


Figure 4
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  		FIGURE 4.
Suppression of AnV protein expression in growth plate chondrocytes using siRNA. Growth plate chondrocytes were transfected with siRNA specific for annexin V (siAnV). Annexin V protein expression was determined after a 3-day infection. Annexin V protein levels were reduced by ~70% when compared with uninfected cells. To test for specificity and toxicity, blots were immunostained with antibodies specific for actin. Note the suppression of annexin V protein expression in siAnV-transfected growth plate cells when compared with the expression level in untransfected cells (Untransfected).

 


Figure 5
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  		FIGURE 5.
Cell viability evaluated by MTT assay and caspase-3 activity of untreated (Control), Pen-SDNRYIGSW-treated (Pen-SDNRYIGSW), Pen-VVISYSMPD-treated (Pen-VVISYSMPD), annexin V-specific siRNA-transfected (siAnV), siAnV-transfected and Pen-SDNRYIGSW-treated (Pen-SDNRYIGSW/siAnV), and siAnV-transfected and Pen-VVISYSMPD-treated (Pen-VVISYSMPD/siAnV) growth plate chondrocytes. Pen-VVISYSMPD treatment reduced cell viability (A) and increased caspase-3 activity (B) when compared with the levels in Pen-SDNRYIGSW-treated cells. However, annexin V-specific siRNA inhibited the effects of Pen-VVISYSMPD on cell viability (A) and caspase-3 activity (B). Data were obtained from three different cultures and were expressed as mean ± S.D. (Pen-VVISYSMPD versus control; *, p ≤ 0.01).

 
Next, we tested whether PKC regulation by annexin V/beta5 integrin interactions is specific for PKC{alpha}. We treated growth plate chondrocytes with Pen-VVISYSMPD and Myr-PKC{alpha}/beta (specific for PKC{alpha}) or Myr-PKC{zeta} (specific for PKC {zeta}). In the absence of Pen-VVISYSMPD, Myr-PKC{alpha}/beta did not change cell morphology and viability when compared with the morphology and viability of untreated cultures (Fig. 7, A, Untreated, Myr-PKC{alpha},beta, and B, white bars, Untreated, Myr-PKC{alpha},beta). However, Myr-PKC{zeta} treatment in the absence of Pen-VVISYSMPD resulted in a notable decrease of growth plate chondrocyte viability (Fig. 7B, white bars, Myr-PKC{zeta}). As already shown above, treatment of growth plate chondrocytes with Pen-VVISYSMPD resulted in a morphological appearance of dead cells (Fig. 7A, Pen-VVISYSMPD) and a marked decrease of growth plate chondrocyte viability when compared with untreated cells (Fig. 7B). Pen-SDNRYIGSW-treated cells showed a similar morphology as untreated cells (Fig. 7A, Untreated, Pen-SDNRYIGSW). Myr-PKC{alpha}/beta and Pen-VVISYSMPD-treated growth plate chondrocytes had a morphological appearance similar to untreated cells (Fig. 7A, Untreated, Pen-VVISYSMPD/Myr-PKC{alpha},beta). In addition, Myr-PKC{alpha}/beta led to an increase of cell viability of Pen-VVISYSMPD-treated growth plate chondrocytes (Fig. 7B, black bars, Myr-PKC{alpha},beta). Myr-PKC{zeta} resulted in a further decrease of cell viability of Pen-VVISYSMPD-treated growth plate chondrocytes (Fig. 7B, black bars, Myr-PKC{zeta}).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we provide evidence that the balance between annexin V/beta5 integrin and annexin V/PKC{alpha} interactions plays a role in the regulation of growth plate chondrocyte apoptosis. Apoptosis is the final fate of terminally differentiated growth plate chondrocytes and is required for normal endochondral bone formation (2, 21). Disturbance of apoptosis in growth plate cartilage results in abnormal bone development. For example, bcl-2 knock-out mice show accelerated chondrocyte differentiation and apoptosis, resulting in accelerated endochondral bone formation and short stature of these mice (6). Our results show that a peptide mimic of annexin V (Pen-VVISYSMPD) that binds to beta5 integrin but not to PKC{alpha} (11) stimulates apoptotic events in hypertrophic growth plate chondrocytes. Similarly, overexpression of annexin V in hypertrophic growth plate chondrocytes increased caspase-3 activity and the proapoptotic bax gene expression and decreased expression of the antiapoptotic bcl-2 gene, suggesting that high expression of annexin V in growth plate chondrocytes results in apoptosis of these cells. On the other hand, suppression of annexin V in hypertrophic growth plate chondrocytes using siRNA resulted in an increase of cell viability and a decrease of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, further confirming our model that both the interactions between beta5 integrin and annexin V (or its peptide mimic) and the interactions between annexin V and PKC{alpha} are required for the regulation of growth plate chondrocyte apoptosis.

Annexin V and beta5 integrin are expressed by hypertrophic and terminally differentiated chondrocytes in growth plate cartilage (13, 14). Furthermore, we have previously shown that retinoic acid-induced terminal differentiation and apoptotic events in growth plate chondrocytes are accompanied by stimulation of annexin V, annexin II, and annexin VI expression (3, 4). How does annexin V regulate terminal differentiation events and apoptosis of growth plate chondrocytes? Apoptotic events are regulated by the interactions of a variety of pathways, including alterations of cytosolic Ca2+ homeostasis. Annexins II, V, and VI form Ca2+ channels in the plasma membrane of terminally differentiated growth plate chondrocytes, leading to the influx of extracellular Ca2+ into these cells. These increases in cytoplasmic Ca2+ stimulate a whole series of events, including stimulation of expression of terminal differentiation and mineralization-related marker genes, release of mineralization-competent matrix vesicles, and apoptotic-related events (4). The present study shows that annexin V regulates apoptosis not only by forming Ca2+ channels but also through its interactions with beta5 integrin and PKC{alpha}. Both events together are required for effective regulation of apoptosis (Fig. 8). The inhibition of one pathway results in an only partial inhibition of cell death (Figs. 5, 6 and 7) (see also Ref. 4). Although the peptide mimic of annexin V only stimulates the beta5 integrin/annexin V/PKC{alpha} pathway, overexpression of annexin V stimulates both the cytosolic Ca2+ and the beta5 integrin/annexin V/PKC{alpha} pathways. An inhibitor of PKC (calphostin) decreased caspase-3 activity and increased cell viability of growth plate chondrocytes treated with Pen-VVISYSMPD, whereas treatment of cells with Pen-VVISYSMPD and an activator of PKC (PMA) further increased caspase-3 activity and decreased cell viability when compared with the levels of Pen-VVISYSMPD-treated cells. This effect was specific for PKC{alpha} in that a specific inhibitor of PKC{alpha} (Myr-PKC{alpha}/beta) increased viability of Pen-VVISYSMPD-treated growth plate chondrocytes. A previous study showed that Pen-VVISYSMPD binds to beta5 integrin but not to PKC{alpha}. In contrast, full-length annexin V binds to both beta5 integrin and PKC{alpha} (11). Binding to beta5 integrin competes with binding of annexin V to active PKC{alpha} (11). Furthermore, other studies have shown that annexin V binds only to the active form of PKC{alpha} but not to the inactive form. Binding of annexin V to active PKC{alpha} results in annexin V-specific PKC inhibition (12, 19, 22). Therefore, our data support the following model. In the presence of beta5 integrin and modest amounts of annexin V, most of annexin V is bound tobeta5 integrin and not to PKC{alpha}, resulting in an active PKC{alpha} and signaling events mediated by PKC{alpha} (Fig. 8A). In the presence of high amounts of annexin V, as in terminally differentiated growth plate chondrocytes, sufficient amounts of annexin V are present to bind to both beta5 integrin and active PKC{alpha}, resulting in an annexin V-specific inhibition of PKC{alpha} and its signaling events (Fig. 8B). In the presence of modest amounts of annexin V, annexin V-specific inhibition of PKC{alpha} and subsequent cell death can be mimicked by Pen-VVISYSMPD, which binds to beta5 integrin but not PKC{alpha}. The interaction of Pen-VVISYSMPD with beta5 integrin releases annexin V from beta5 integrin binding, allowing annexin V to bind to active PKC{alpha} (Fig. 8B). The presence of an activator of PKC{alpha} (PMA) further decreased cell viability of Pen-VVISYSMPD-treated growth plate chondrocytes because PMA has been shown to facilitate binding of annexin V to active PKC{alpha} (Fig. 8C) (see also Ref. 11). On the other hand, inhibitors of PKC{alpha} prevent binding of annexin V to PKC{alpha} in the presence of high amounts of annexin V or Pen-VVISYSMPD, thereby hindering annexin V-specific inhibition of PKC{alpha} and subsequent cell death (Fig. 8D).


Figure 6
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  		FIGURE 6.
Cell viability and caspase-3 activity of untreated (Control), Pen-SDNRYIGSW-treated (Pen-SDNRYIGSW), Pen-VVISYSMPD-treated (Pen-VVISYSMPD), PMA-treated (PMA), calphostin-treated (Calphostin), Pen-SDNRYIGSW- and PMA-treated (Pen-SDNRYIGSW/PMA), Pen-VVISYSMPD- and PMA-treated (Pen-VVISYSMPD/PMA), Pen-SDNRYIGSW- and calphostin-treated (Pen-SDNRYIGSW/Cal.), and Pen-VVISYSMPD- and calphostin-treated (Pen-VVISYSMPD/Cal.) growth plate chondrocytes. PMA further decreased cell viability (A) and further increased caspase-3 activity (B) in Pen-VVISYSMPD-treated cells. In contrast, calphostin increased cell viability (A) and decreased caspase-3 activity (B) in Pen-VVISYSMPD-treated cells. Data were obtained from three different cultures and expressed as mean ± S.D. (Pen-VVISYSMPD or Pen-VVISYSMPD/PMA versus control; *, p ≤ 0.01).

 
Interestingly, active PKC{alpha} has been implicated in mediating cell survival in a variety of cell types (23, 24). Our findings that suppression of annexin V expression in growth plate chondrocytes increased cell viability and decreased caspase-3 activity in Pen-VVISYSMPD-treated cells suggest that active PKC{alpha} also mediates cell survival in chondrocytes and that inhibition of PKC{alpha} by annexin V and/or the interactions among annexin V, beta5 integrin, and PKC{alpha} are required for the stimulation of apoptotic events in growth plate chondrocytes. Inactivation of PKC{alpha} has been shown to play an important role in modulating hepatic apoptosis during sepsis. Apoptosis of hepatocytes after sepsis is associated with a decrease in bcl-2 expression and an increase in bax expression, which is similar to the findings of our study in which PKC{alpha} in growth plate chondrocytes was inhibited by annexin V, resulting in decreased expression of bcl-2 and increased expression of bax (23). Other findings have shown that PKC{alpha} inhibition in NIH3T3 cells reduced proliferation and induced apoptosis and that these effects were also mediated by alterations of bcl-2 and bax expression (25). Similar to the findings of our study, a recent study showed that Pen-VVISYSMPD mediates apoptosis of endothelial cells by a similar PKC{alpha}/beta5integrin/annexin V-mediated mechanism (11). Therefore, it is plausible to assume that PKC{alpha}/annexin V/beta5 integrin interactions play a role not only in the regulation of growth plate chondrocyte apoptosis but also in the regulation of apoptosis of other cell types.


Figure 7
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  		FIGURE 7.
A, light microscopical analysis revealed a morphology of dead cells in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas Myr-PKC{alpha}/beta resulted in a morphology of Pen-VVISYSMPD-treated growth plate chondrocytes similar to untreated, Pen-SDNRYIGSW, or Myr-PKC{alpha}/beta-treated growth plate chondrocytes. B, quantification of cell viability of control, Myr-PKC{alpha}/beta-treated, or Myr-PKC{zeta}-treated growth plate chondrocytes in the absence (white bars; -Pen-VVISYSMPD) or presence (black bars; +Pen-VVISYSMPD) of Pen-VVISYSMPD. Cell viability was measured using the MTT assay after 24 h of treatment. Data were obtained from three different cultures and expressed as mean ± S.D. (control + Pen-VVISYSMPD versus control - Pen-VVISYSMPD; Myr-PKC{zeta} - Pen-VVISYSMPD versus control - Pen-VVISYSMPD; Myr/PKC{zeta} + Pen-VVISYSMPD versus control - Pen-VVISYSMPD; *, p ≤ 0.01).

 
PKCs have been shown to play important roles in cell differentiation and to act as either proapoptotic or antiapoptotic depending on the cell type, extracellular stimuli, and specific isoform that is activated or inhibited. Our findings showing that the interactions among annexin V, beta5 integrin, and active PKC{alpha} play a proapoptotic role in growth plate chondrocytes suggest that not only activation or inhibition of PKC but also the mechanism of PKC activation or inhibition plays a role in whether PKCs act pro- or antiapoptotic. Although the interactions among annexin V, beta5 integrin, and active PKC{alpha} were able to affect cell survival of growth plate chondrocytes, a specific inhibitor of PKC{zeta} (Myr-PKC{zeta}) decreased cell survival of untreated and Pen-VVISYSMPD-treated growth plate chondrocytes, suggesting that PKC{zeta} plays an additional role in cell survival of hypertrophic growth plate chondrocytes independent of the role of beta5 integrin/annexin V/PKC{alpha} interactions. These findings are consistent with previous findings showing that inhibition of PKC{zeta} is required for nitric oxide-induced apoptosis of articular chondrocytes and that inhibition of PKC{zeta} activity is required for the apoptosis of various cell types (20, 26, 27). Therefore, it is possible that the regulation of the activities of various PKCs by different mechanisms plays a crucial role in the regulation of growth plate and articular chondrocyte apoptosis.

Bcl-2 is expressed in chondrocytes throughout the growth plate, with highest levels in late proliferative and prehypertrophic chondrocytes and markedly decreased levels in terminally differentiated chondrocytes. The opposite pattern was observed for Bax expression, with undetectable levels in proliferative cells and a progressive increase toward hypertrophic chondrocytes. Thus, within the growth plate, the ratio of Bcl-2 to bax progressively decreases in chondrocytes in favor of Bax (6). This change in the bcl-2:bax ratio in favor of bax results in the apoptotic death of terminally differentiated chondrocytes (1, 2, 5). Our study findings show that annexin V/beta5 integrin/PKC{alpha} interactions result in the alteration of bcl-2 and bax expression in favor of bax, suggesting that annexin V/beta5 integrin/PKC{alpha} interactions may affect apoptosis of growth plate chondrocytes by altering the bcl-2:bax ratio in favor of bax. We and others have shown that articular chondrocytes in osteoarthritic cartilage undergo differentiation events similar to those of growth plate chondrocytes, resulting in terminal differentiation of osteoarthritic chondrocytes (13, 28, 29). Interestingly, these cells also express annexin V and beta5 integrin (13, 30). Furthermore, several studies have shown that chondrocytes in osteoarthritic or damaged cartilage undergo apoptotic changes (13, 31-34). Therefore, it is possible that annexin V, beta5 integrin, and PKC{alpha} are involved in the regulation of apoptosis of articular chondrocytes in osteoarthritis by a mechanism similar to that described in the present study for growth plate chondrocytes. Interestingly, previously it has been reported that nitric oxide-induced apoptosis of articular chondrocytes requires the inhibition of PKC{alpha} and -{zeta} consistent with the findings of our study (20).


Figure 8
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  		FIGURE 8.
A, in the presence of modest amounts of AnV, such as in prehypertrophic and hypertrophic growth plate chondrocytes, most of AnV is bound to beta5 integrin (beta5) but not to active PKC{alpha} ({alpha}). Active PKC{alpha} mediates cell survival signals. An, annexin II, V, and VI. B, in the presence of high amounts of AnV, such as in terminally differentiated growth plate chondrocytes, or in the presence of modest amounts of AnV and the annexin V peptide mimic Pen-VVISYSMPD (V), Pen-VVISYSMPD or some annexin V binds to beta5 integrin, whereas the remaining AnV binds to and inhibits active PKC{alpha}. The annexin V-specific inhibition of PKC{alpha} leads to cell death. In addition, the annexin II, V, and VI-mediated influx of Ca2+ in growth plate chondrocytes also contributes to cell death. C, the presence of PMA facilitates the binding of AnV to active PKC{alpha}, resulting in increased cell death in Pen-VVISYSMPD-treated or terminally differentiated (high amounts of annexin V-expressing) growth plate chondrocytes. D, inhibitors of PKC{alpha} (calphostin, Myr-PKC{alpha}/beta) prevent binding of AnV to PKC{alpha} and annexin V-specific inhibition of PKC{alpha} and ultimately cell death.

 
beta5 integrin is expressed by various skeletal cell types. A previous study detected high expression of beta5 integrin in developing cartilages and suggested that beta5 integrin is a key integrin involved in bone formation (35). However, mice lacking beta5 integrin show no obvious skeletal phenotype (36). It is possible that other integrins, such as beta3 integrin, might compensate for the loss of beta5 integrin. Sometimes a lack of phenotype in mice can also result from other reasons, including the location of the neomycin cassette and/or the genetic background of the mice. For example, recently two different lines of annexin VII-/- mice were generated; one mouse line shows a severe phenotype, whereas the other mouse line has only a mild phenotype (37, 38).

In conclusion, we present evidence that the link among annexin V, beta5 integrin, and PKC{alpha} interactions mediates regulation of apoptosis of growth plate chondrocytes. Our findings reveal that a certain amount of annexin V is required to interact with beta5 integrin and PKC{alpha} and that the interactions of annexin V with both beta5 integrin and PKC{alpha} are required for the induction of apoptosis of growth plate chondrocytes. Increasing the amounts of annexin V present in growth plate chondrocytes to interact with beta5 integrin and/or PKC{alpha} either by overexpression of annexin V or by adding the annexin V-mimicking peptide (Pen-VVISYSMPD) stimulated apoptotic events in growth plate chondrocytes, whereas suppressing annexin V expression prevented apoptotic events in growth plate chondrocytes even in the presence of the annexin V peptide mimic Pen-VVISYSMPD. Endothelial cells seem to undergo apoptosis by a similar mechanism involving annexin V/beta5 integrin and PKC (11). Considering that annexin V and beta5 integrin are also expressed in osteoarthritic articular cartilage (13, 30, 39) and that inhibition of PKC{alpha} seems to be involved in articular chondrocyte apoptosis (20), it is possible that a similar mechanism leads to cell death of osteoarthritic chondrocytes.


   FOOTNOTES
 
* This work was supported by grants from the NIAMS National Institutes of Health Grants AR049074 and AR046245 (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Orthopedics, University of Maryland School of Medicine, 20 Penn St. HSFII S003D, Baltimore, MD 21201. Tel.: 410-706-2417; Fax: 410-706-0028; E-mail: tkirsch{at}umoa.umm.edu.

2 The abbreviations used are: PKC, protein kinase C; Pen, Penetratin; PMA, phorbol 12-myristate 13-acetate; Myr, myristoylated; RCAS-BP, Rous sarcoma virus-based expression vector; siRNA, small interfering RNA; Bis-Tris, 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-dioll; AnV, annexin V; siAnV, siRNA specific for AnV; DAPI, 4',6-diamidino-2-phenylindole; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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