Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells

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Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells Cyril Thouverey a,b,c,d,e,f , Agata Malinowska g , Marcin Balcerzak a , Agnieszka Strzelecka-Kiliszek a , René Buchet b,c,d,e,f , Michal Dadlez g,h , Slawomir Pikula a,⁎ a

Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland Université de Lyon, Lyon, F-69361, France c Université Lyon 1, Villeurbanne, F-69622, France d INSA-Lyon, Villeurbanne, F-69622, France e CPE Lyon, Villeurbanne, F-69616, France f ICBMS CNRS UMR 5246, Villeurbanne, F-69622, France g Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinski St., 02-106 Warsaw, Poland h Institute of Genetics and Biotechnology, Department of Biology, Warsaw University, 5A Pawinski St., 02–106 Warsaw, Poland b

AR TIC LE I N FO

ABS TR ACT

Article history:

Matrix vesicles (MVs), released by budding from apical microvilli of osteoblasts during bone

Received 24 February 2011

formation and development, are involved in the initiation of mineralization by promoting

Accepted 8 April 2011

the formation of hydroxyapatite in their lumen. To gain additional insights into MV

Available online 15 April 2011

biogenesis and functions, MVs and apical microvilli were co-isolated from mineralizing osteoblast-like Saos-2 cells and their proteomes were characterized using LC–ESI-MS/MS

Keywords:

and compared. In total, 282 MV and 451 microvillar proteins were identified. Of those, 262

Apical microvilli

were common in both preparations, confirming that MVs originate from apical microvilli.

LC–ESI-MS/MS

The occurrence of vesicular trafficking molecules (e.g. Rab proteins) and of the on-site protein

Matrix vesicles

synthetic machinery suggests that cell polarization and apical targeting are required for the

Mineralization

incorporation of specific lipids and proteins at the site of MV formation. MV release from

Osteoblasts

microvilli may be driven by actions of actin-severing proteins (gelsolin, cofilin 1) and contractile motor proteins (myosins). In addition to the already known proteins involved in MV-mediated mineralization, new MV residents were detected, such as inorganic pyrophosphatase 1, SLC4A7 sodium bicarbonate cotransporter or sphingomyelin phosphodiesterase 3, providing additional insights into MV functions. © 2011 Elsevier B.V. All rights reserved.

Abbreviations: AR-S, Alizarin Red-S; ECM, extracellular matrix; FPR, false positive rate; GPI, glycosylphosphatidyl inositol; HA, hydroxyapatite; HBSS, Hank's balanced salt solution; MVs, matrix vesicles; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; PS, phosphatidylserine. ⁎ Corresponding author. Tel.: +48 22 589 2347; fax: + 48 22 822 5342. E-mail address: [email protected] (S. Pikula). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.04.005

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1.

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Introduction

During skeletogenesis and bone development, osteoblasts from bones as well as hypertrophic chondrocytes from embryonic or growth plate cartilages, regulate the synthesis of extracellular matrix (ECM) proteins and then initiate matrix mineralization by releasing matrix vesicles (MVs) [1–3]. Mineralizing cells were shown to concentrate inorganic phosphate (Pi) in their cytoplasm [4] as well as high levels of Ca2+ ions in their mitochondria [5] prior to mineralization. Mitochondrial Ca2+ ions are released and, in combination with high levels of Pi, form amorphous calcium phosphate at sites of MV formation [6]. MVs are released from apical microvilli of hypertrophic chondrocytes [3,7] or osteoblasts [8] into the ECM by budding and pinching off. Once released, MVs continue to accumulate Ca2+ ions and Pi which promote hydroxyapatite (HA) formation from the immature minerals present in their lumen [9]. The second phase of mineralization starts with the release of HA crystals from MVs and the propagation of mineral formation in the ECM [3]. MVs are round membranous structures with a diameter ranging from 100 to 400 nm. MV membrane, which is enriched in cholesterol, sphingomyelin and phosphatidylserine, has a lipid composition very similar to microvillar membrane of parent cells [7,8]. MVs and microvilli have similar protein profiles with analogous enrichment in TNAP, Na+/K+-ATPase, AnxA2 and AnxA6. MVs and microvilli contain actin and actinregulating proteins [10–12], and retraction of the supporting actin microfilament network is critical for MV budding and release [7,8]. Cofilin-1, an actin-severing protein, has been suggested to participate in this process since it co-localizes with MV markers in apical microvilli of mineralizing osteoblastlike cells [8]. This network of cytoskeletal proteins not only facilitates MV formation, but may also support their shape and mobility within the ECM [10]. MVs perform specialized functions essential for the initial step of mineral formation. These functions include control of Ca2+ and Pi ion homeostasis, mineral nucleation, hydrolysis of mineralization inhibitors such as inorganic pyrophosphate (PPi), modification of the surrounding matrix, maintenance of membrane lipid composition, and interactions with ECM components controlling mineral growth and localization [3,13,14]. MVs possess a protein and lipid machinery essential to execute these functions. MVs contain different proteins regulating Pi and PPi homeostasis. Among them, tissue non-specific alkaline phosphatase (TNAP) is a GPI-anchored enzyme providing extravesicular Pi by hydrolyzing a variety of organic phosphate esters including PPi [15]. Ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is a transmembrane enzyme producing extravesicular PPi [16], a potent inhibitor of HA growth [17,18], from nucleotide triphosphate hydrolysis. NPP1 and TNAP have antagonistic effects on mineral formation due to their opposing activities [19]. 5′-nucleotidase is also a GPIanchored enzyme delivering extravesicular Pi by hydrolyzing AMP [20]. Ion-motive ATPases [20] and PHOSPHO1 [21] are involved in the production of intravesicular Pi. Ca2+-ATPase, Na+/K+-ATPase [10] and vacuolar proton pump ATPase [11] can hydrolyze ATP yielding Pi for mineralization. PHOSPHO1 is a

luminal enzyme providing Pi for mineralization from phosphocholine and phosphoethanolamine hydrolysis. Pi transport from ECM to the MV lumen may be performed by a sodium-dependent Pi transporter (a member of the type III Glvr-1 gene family) [22] or by an alkaline pH-specific Pi transporter which is not strictly sodium-dependent [23]. Several annexins (AnxA1, AnxA2, AnxA4, AnxA5, AnxA6, AnxA7 and AnxA11) were identified in MVs [10,11]. Annexins are Ca2+- and phospholipid-binding proteins which, under the right conditions, can form Ca2+ channels through MV membranes [24,25]. Therefore, annexins are thought to mediate Ca2+ influx into MVs [25]. The inner leaflet of the MV membrane, enriched in anionic phospholipids such as phosphatidylserine (PS) [8,26,27], is involved in the formation of the initial mineral phase in developing bone. MVs contain a PS-associated pool of Ca2+, Pi, and annexins that constitute a nucleation complex allowing mineral transformation from the amorphous to crystalline phase [28]. The pH is critical for mineralization since acidic pH inhibits mineral deposition. Therefore, intravesicular pH may be regulated by carbonic anhydrase II [29]. Phospholipase A2 [30] and phospholipase D [31] activities have been found to influence MV membrane lipid composition and to regulate MV membrane breakdown and, thus, mineral release within the ECM. Extracellular matrix proteins such as collagens [10,11,25] and proteoglycans [32] interact with their receptors such as AnxA5 [25] or integrins [10,11] present at the MV membrane. These interactions between ECM components and MVs are necessary to control mineral growth and its directional expansion [3]. Furthermore, several enzymes such as matrix metalloproteases [33,34], carboxypeptidase M [11] and aminopeptidases [10] are present at the outer surface of MVs. They catalyze degradation of the ECM, hydrolyze proteins that inhibit mineralization and, thus, increase MV membrane permeability to ions and the access of MVs to sites of mineralization [33,34]. In addition, ECM degradation promotes the release of osteogenic factors such as bone morphogenetic proteins and transforming growth factor β and therefore actively influences surrounding osteoblasts [34]. To delineate the molecular mechanisms of MV biogenesis and release and to obtain additional insights into MV functions, MVs and apical microvilli were co-isolated from mineralizing osteoblast-like Saos-2 cells and their respective protein composition determined by proteomic analyses using LC–ESI-MS/MS. The possible mechanisms supporting MVs formation are discussed in confrontation with data concerning the biogenesis and function of microvilli. Finally, the possible roles of newly identified proteins in MV-mediated mineralization are proposed.

2.

Materials and methods

2.1.

Mineralizing Saos-2 cell cultures

Human osteosarcoma Saos-2 cells (ATCC HTB-85) were cultured in McCoy's 5A (ATCC) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (both from Sigma) and 15%

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(v:v) fetal bovine serum (FBS) (Gibco). Mineralization was induced by culturing the confluent cells in growth medium supplemented with 50 μg/mL ascorbic acid (Sigma) and 7.5 mM β-glycerophosphate (Sigma). Mineralization in Saos-2 cell cultures was assessed by Alizarin Red-S (AR-S) staining as described in [8].

2.2.

Isolation of microvilli and matrix vesicles

Microvilli and MVs were co-isolated from mineralizing Saos-2 cell cultures as previously described [8]. Saos-2 cell cultures were digested with 100 U/mL collagenase Type IA (Sigma) in Hank's balanced salt solution at 37 °C for 3 h (digest). Then, cells were centrifuged (600 × g, 15 min) yielding pellet 1. The obtained supernatant was centrifuged (20,000 × g, 20 min) to sediment all the cell debris, nuclei, mitochondria, lysosomes in pellet 2. The second supernatant was subjected to an ultracentrifugation (80,000 × g, 60 min) yielding pellet 3 containing MVs. In parallel, pellet 1 containing intact cells, was homogenized in 5 mL of sucrose buffer in the presence of protease inhibitor cocktail (Sigma). The homogenate was then centrifuged twice at 10,000 × g for 15 min to sediment intact cells, cell debris, nuclei, mitochondria, lysosomes (pellet A). To separate the microvillar membranes from the basolateral plasma membranes, supernatant A was supplemented with 12 mM MgCl2, stirred at 4 °C for 20 min to induce basolateral membrane precipitation and centrifuged twice at 2,500 × g for 10 min to pellet aggregates of basolateral membranes (pellet B). The supernatant was centrifuged at 12,000 × g for 60 min to pellet microvilli (pellet C). The last supernatant contained microsomal and cytosolic fractions. Fractions containing MVs or microvilli (pellet 3 and pellet C, respect ively) (8.6% sucrose, w:v) were overlaid on a sucrose step gradient made with 45%/37%/25% (w:v) sucrose and centrifuged at 90,000 × g for 5 h. The 25% and 37% sucrose fractions were collected, pooled, diluted ten-fold in HBSS and centrifuged at 120,000 × g for 60 min. Protein concentration in the fractions was determined using the Bio-Rad Protein Assay. The integrity of MVs was evaluated using electron microscopy [8]. The ability of MVs to mineralize was assessed by analyzing the composition of MV-produced minerals by the Fourier transform infrared spectroscopy [8].

2.3.

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activity was assayed at 37 °C using p-nitrophenyl phosphate as substrate in piperazine glycyl–glycine buffer at pH 5 with 0.2% Triton X-100 (v:v). Enzyme activity is described as ΔA420 per hour per mg of proteins. Leucine aminopeptidase activity was determined at 37 °C using L-leucine-p-nitroanilide as substrate in phosphate buffer at pH 7.4 by recording absorbance at 410 nm (εp-nitroanilide = 8.8 mM− 1∙cm− 1). Enzyme activity is quantified in μmol of p-nitroanilide released per hour per mg of proteins. TNAP activity was measured using p-nitrophenyl phosphate as substrate at pH 10.4 by recording absorbance at 420 nm (εp-NP is equal to 18.8 mM− 1∙cm− 1). Enzyme unit is defined as μmol of p-nitrophenolate released per min per mg of proteins.

2.4. Tandem mass spectrometry analysis of protein digests MV and microvillar proteins were prepared for MS analysis using two methods. First, equal amounts of MV and microvillar proteins (20 μg) were boiled in Laemmli gel loading buffer at 100 °C for 5 min, separated on one dimensional SDS(10%, w/v) polyacrylamide gels, and stained with Coomassie Brilliant Blue (Sigma). Each gel was cut into eleven slices (as indicated in Fig. 1) which were then submitted to a standard in-gel tryptic digestion [35]. Second, MV and microvillar samples containing equal amounts of proteins (20 μg) were directly digested and alkylated in solution without a prior SDS-PAGE step, to ensure that all proteins, including the hydrophobic and low-molecular-weight ones, were analyzed. Samples were supplemented with 10 ng/μL trypsin (Promega) in 25 mM ammonium hydrocarbonate (pH 8.5), and incubated overnight at 37 °C. The samples were then incubated with

Assay of marker enzyme activities

To assess the purity of the isolated microvilli and MVs, different marker-enzyme activities were measured in the different fractions obtained from microvilli and MV preparations. Succinate dehydrogenase activity was determined spectrophotometrically (λ = 630 nm) at 37 °C in 0.5 mL of phosphate buffer containing 0.05% (w:v) nitroblue tetrazolium, 0.2% (v:v) Triton X-100, 20 mM succinate sodium, pH 7.4. Enzyme activity is described as change in absorbance (ΔA630) per hour per mg of proteins. NADH oxidase activity was determined at 37 °C in 1 mL containing 0.14 mM NADH, 1.3 mM potassium ferricyanide, 10 mM Tris–HCl, pH 7.5 by recording absorbance at 340 nm (εNADH = 6.22 mM− 1∙cm− 1). Enzyme activity is quantified in μmol of NADH oxidized per hour per mg of proteins. Acid phosphatase

Fig. 1 – SDS-PAGE of Saos-2 cell microvilli and MVs. Coomassie-brilliant-blue stained 10% SDS-PAGE of microvilli and matrix vesicles (MVs). 11 gel slices were cut (as shown) for in-gel tryptic digestion and protein identification by tandem mass spectrometry.

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10 mM DTT for 30 min at 56 °C to reduce cysteins, and then alkylated with 50 mM iodoacetamide for 45 min in the darkness at room temperature. The reaction was stopped with the addition of 0.1% TFA (v/v) and samples were stored at 4 °C. Equal volumes of the tryptic peptide mixtures were applied to a RP-18 precolumn (Waters) using a 0.1% (v/v) TFA solution as the mobile phase, and transferred to a nano-HPLC RP-18 column (Waters, length: 250 mm, bead diameter: 1.7 μm). Peptide mixtures were separated using an ACN gradient (0–30% ACN in 45 min) in the presence of 0.1% (v/v) formic acid at a flow rate of 250 nL/min. The column was connected to the ESI (Finningan Nanospray, Thermo) ion source of a LTQ-FTICR mass spectrometer (Thermo) working in the regime of data-dependent MS to MS/MS switch. A blank run ensuring lack of cross contamination from previous samples preceded each analysis. Three separate runs were carried out for each sample. In the first, the spectrometer fragmented the ten most intense peaks in each cycle. In the second run, the ten most intense peaks were omitted and the next ten most intense peaks were fragmented. In the third run, the twenty most intense peaks were omitted and the next ten most intense peaks were fragmented. Data resulting from the three experiments were merged into a single database search file using a “merge MS/MS data into a single file” option of the MASCOT Daemon program. Proteomic analyses were performed on 3 independent preparations of microvilli and MVs.

2.5.

3.

Results

3.1. Characteristics of matrix vesicles and apical microvilli from Saos-2 cell cultures Human osteosarcoma Saos-2 cells undergo the entire osteoblastic differentiation program from proliferation to mineralization [36]. During their differentiation, Saos-2 cells acquire a polar shape and start to release MVs from their apical microvilli [8]. Therefore, Saos-2 cell cultures were selected, as a convenient model for osteoblastic mineralization, to analyze the biogenesis and functions of MVs. Saos-2 cells were cultured in the presence of ascorbic acid and β-glycerophosphate, two osteogenic factors known to stimulate osteoblastic differentiation and mineralization [37]. After the differentiation period, apparent calcium nodules characteristic of osteoblastic mineralization were detected by AR-S staining (Fig. 2 A–B). Stimulated Saos-2

Database searching

Obtained mass spectra were preprocessed with the Mascot Distiller software (v. 2.2.1, Matrix Science) and searched against the latest version of non-redundant protein database from the NCBI (NCBInr, 5633163 sequences, 1947344958 residues), using the engine 8-processor on-site licensed MASCOT software (Mascot Server v. 2.2.03, Mascot Daemon v. 2.2.2, Matrix Science). Search parameters were set as follows: taxonomy: human (197177 sequences); enzyme: semiTrypsin; fixed modifications: carbamidomethylation (C); variable modifications: carbamidomethylation (K), oxidation (M); protein mass: unrestricted; peptide mass tolerance: ±40 ppm; MS/MS fragment ion mass tolerance: ±0.8 Da; max missed cleavages: 1. To confirm the statistical validity of protein hits, the false positive rate (FPR) values were calculated. All mass spectra were searched against a randomized NCBI database using search settings as above, and the FPR was computed by dividing the number of accepted queries from the randomized search by the number of accepted queries from the standard search, and multiplying the result by 100%. Mascot score threshold used for acceptance of peptide identification was calculated to keep FPR close to 1%. Only peptides with Mascot score values above 50 (for solutions, corresponding to FPR 1.08%) and 54 (for gel slices, corresponding to 1.03%) were accepted. Only protein hits characterized by at least two high-scoring peptides were accepted. All identified proteins were searched against the Human Protein Reference Database (http://www.hprd.org) to determine their classification, molecular weight, number of transmembrane domains and post-translational modifications, and subcellular localization.

Fig. 2 – Mineralization by Saos-2 cells. (A) Saos-2 cells were incubated for 6 days in the absence (control) or presence of 50 μg/mL AA and 7.5 mM β-GP (stimulation), stained with AR-S to detect calcium nodules and visualized under an Axio Observer.Z1 fluorescence microscope (Zeiss) using transmitted light and phase contrast filter. (B) Saos-2 cells were cultured 6 days under normal conditions or stimulated, stained with AR-S and photographed. (C) AR-S was solubilized in control and stimulated cell cultures by cetylpyridinium chloride and quantified at 562 nm (Results are presented in μmol of AR-S per culture and are mean ± SD, n = 3).

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cells produced 6 times more calcium minerals than resting Saos-2 cells within 6 days (Fig. 2C). MVs were isolated from mineralizing Saos-2 cell cultures. To evaluate the integrity of MVs, they were examined using electron microscopy. MVs were recognized as spherical vesicle structures with a diameter ranging from 100 to 400 nm, delimited by a characteristic trilaminar membrane (Fig. 3A). In some cases, it was possible to observe needle-like electron-dense mineral deposits inside the MVs (Fig. 3B), showing their ability to mineralize. Moreover, after 6 h of incubation in a mineralization buffer containing 2 mM Ca2+ and 3.42 mM Pi, MVs were able to form HA minerals as proved by examination of the infrared spectrum of the mineral deposit which was identical to the spectrum of the HA standard (Fig. 3C). Apical microvilli were isolated from mineralizing Saos-2 cells. To assess the purity of the isolated microvilli and MVs, different marker-enzyme activities were determined (Table 1). Microvilli and MV preparations exhibited either none or only trace activities of succinate dehydrogenase (inner mitochondrial membrane marker), of NADH oxidase (mitochondrial and endoplasmic reticulum marker) and of acid phosphatase (lysosome marker) (Table 1), indicating their relative purity. Moreover, leucine aminopeptidase (apical membrane marker enzyme) and TNAP (MV marker enzyme) activities were highly enriched in both microvilli and MVs (Table 1).

3.2.

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Among them, cytoskeletal proteins [10,12], proteins involved in Pi homeostasis (TNAP [15], NPP1 [16], Na+/K+-ATPase [10,20], plasma membrane Ca2+-ATPase [10,20] and PHOSPHO1 [21]), proteins involved in Ca2+ ion homeostasis (AnxA1, AnxA2, AnxA4, AnxA5, AnxA6, AnxA7, and AnxA11 [10–12]), MV-ECM adhesion proteins (various integrins and proteoglycans such as hyaluronan and proteoglycan link protein 1 [10–12]), and other proteins such as caveolin 1 [42], lactate dehydrogenase B [43] or protein kinase C [44] (Table 3). Novel proteins such as inorganic pyrophosphatase 1, sphingomyelin phosphodiesterase 3, sorcin or SLC4A7 (sodium bicarbonate cotransporter), thought to be involved in MV functions were also identified in MVs (Table 3).

4.

Discussion

4.1. Comparative analysis of microvillar and MV proteomes Among the 451 microvillar and 282 MV proteins, 262 were common to both fractions (Table 2). Therefore, 93% of MV

Microvillar and MV proteomes

The proteomic analyses identified 451 microvillar proteins (Supplemental Table 1) and 282 MV proteins (Supplemental Table 2). The protein profile of microvilli and MVs could be divided into various functional categories (Table 2). The peptide sequence and score, the number of peptide matches as well as the molecular weight, the number of transmembrane domains, the type of post-translational modifications and the subcellular localization of each identified protein are given in Supplemental Tables 1 and 2. Among the 451 proteins detected in Saos-2 cell microvilli, 182 were previously identified in the microvilli from different cell types [38–41] (Supplemental Table 1). Most of these proteins are cytoskeletal components implicated in the maintenance and the motility of microvilli, such as β-actin, myosin 9, actinin α1 and α4, ezrin, moesin, and radixin; Rasrelated proteins Rab1A/B, Rab 5A, Rab7 and Rab14 involved in intracellular trafficking; members of the 14-3-3 family which are signaling proteins; calcium regulating annexins (AnxA1, AnxA2, AnxA4-6, and AnxA11); and other proteins such as leucine aminopeptidase, alkaline phosphatase, enzymes involved in glycolysis (e.g. phosphoglycerate kinase 1, enolase 1, and lactate dehydrogenase), creatine kinase B, Na+/K+-ATPase, 4F2 cell surface antigen and basigin [38–41] (Supplemental Table 1). The MV proteome of mineralizing Saos-2 cells was compared to the three recently described proteomes of MVs isolated from pre-osteoblast MC3T3-E1 cell cultures [10], from chicken embryo growth plate cartilage [11] and from human articular cartilage [12]: 134 proteins were identified in at least one of these studies (Table 2 and supplemental Table 2).

Fig. 3 – Matrix vesicles are the initial sites of hydroxyapatite mineral formation. (A,B) Electron microscopy view of (A) MVs and (B) MV breakdown leading to mineral release. (Magnifications: A, B, ×50,000). (C) Infrared spectra of minerals formed by MVs. MVs were incubated at 37 °C in mineralization buffer for 6 h, then the formed minerals were collected, washed and analyzed by infrared spectroscopy. Infrared spectrum of hydroxyapatite as control (HA) and minerals formed by matrix vesicles (MVs). Infrared spectra representative for three independent measurements are presented.

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proteins were present in microvilli, confirming the purity of our preparation. The remaining 20 MV proteins, which were not detected in microvilli, represented 6 ECM, 6 soluble, 4 membrane-associated and 4 transmembrane proteins (Supplemental Table 2). The main reason why differences were observed is that MVs are natural vesicles released by a physiological process from Saos-2 cells, while microvillar vesicles were artificially obtained. Equal amounts of microvillar and MV proteins were used for proteomic analyses. Therefore, the higher number of proteins identified in microvilli could reflect not only microvillar proteins but also proteins implicated in vesicle formation. For example, spectrins, which are components of the cytoskeletal “terminal web”, were identified in microvilli but not in MVs (Supplemental Tables 1 and 2). MV proteins that play a role in the mineralization process were detected in microvilli as well. Furthermore, a large number of proteins identified in MVs are established marker proteins of microvilli (Supplemental Tables 1 and 2), confirming that MVs originate from the apical microvilli of Saos-2 cells [8]. Different proteins identified in both MVs and microvilli (Table 4), provided new insights into the mechanisms of MV biogenesis as discussed below.

4.2.

Lipid and protein targeting to MVs

MVs and apical microvilli co-isolated from Saos-2 cells exhibited a similar lipid composition and both fractions were particularly rich in cholesterol, sphingomyelin and phosphatidylserine in comparison to the plasma membrane [8]. Among proteins found in both MVs and microvilli, the MS/ MS analysis revealed the presence of GPI-anchored, myristoylated and palmitoylated proteins (Supplemental Tables 1 and 2). Proteins harboring the GPI-anchor target specifically to the sphingolipid- and cholesterol-enriched membrane rafts [45].

Other proteins found in MVs and microvilli in the present work are raft or raft-associated proteins such as flotillin 1 and 2, AnxA2, AnxA6, G proteins, and integrins [46] (Supplemental Tables 1 and 2). These data suggest that MVs may incorporate specific membrane components before budding. Several proteins involved in vesicular fatty acid, cholesterol and phospholipid trafficking (Niemann–Pick C1, copine III, low-density lipoprotein receptor-related protein 1) as well as in vesicular protein trafficking (transmembrane protein trafficking, proteins of the Rab family) were identified in microvilli and MV preparations (Supplemental Tables 1 and 2). Small GTPases belonging to the Rab family such as Rab4, Rab5, Rab11, Rab14, Rab18, and Rab21, which regulate multiple stages of membrane traffic such as vesicle formation, transport and fusion to the apical membrane [47–49], were found (Table 4, Supplemental Tables 1 and 2). Furthermore, components of the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor machinery (vesicleassociated membrane proteins 2 and 7, AnxA2, SNAPα) involved in apical membrane fusion [50,51] were detected in the proteomic analysis (Table 4). Together, the presence of molecules involved in vesicular trafficking may provide evidence for an apical targeting of lipids and proteins from the trans-Golgi network and the endosomal compartments to MV membrane. MVs are also enriched in cytosolic proteins, e.g. PHOSPHO1 or lactate dehydrogenases A and B. The existence of a mechanism for specific uptake of cytosolic lactate dehydrogenases into MVs during their formation has been postulated [43]. The intracellular localization of specific mRNAs is a general mechanism responsible for asymmetric distribution of certain cytoplasmic proteins in polarized cells [52]. This process requires RNA-binding proteins for the recognition of specific mRNAs and for their transport to appropriate

Table 1 – Evaluation of the purity of Saos-2 cell microvillar and matrix vesicle preparations. Cell fraction

Digest

Succinate dehydrogenase Activity 2.12 ± 0.14 % 100 (ΔA630/h/mg protein) E 1 NADH oxidase Activity 13.51 ± 1.18 (μmol oxidized NADH/ % 100 h/mg protein) E 1 Activity 2.26 ± 0.19 Acid phosphatase % 100 (ΔA420/h/mg protein) E 1 Leucine aminopeptidase Activity 1.04 ± 0.12 (μmol p-nitroanilide/ % 100 h/mg protein E 1 Alkaline phosphatase Activity 8.41 ± 0.42 (μmol p-nitrophenolate/ % 100 h/mg protein E 1

Pellet A

Pellet B

4.25 ± 0.79 80.7 ± 15 2 24.37 ± 4.07 72.6 ± 12.1 1.8 4.44 ± 0.29 79 ± 5.2 2 0.74 ± 0.13 28.5 ± 5 0.7 3.2 ± 0.24 15 ± 1.2 0.4

2.33 ± 0.4 8.7 ± 1.5 1.1 20.09 ± 5.80 11.8 ± 3.4 1.5 2.6 ± 0.6 8.7 ± 2.1 1.2 1.23 ± 0.15 9.4 ± 1.2 1.2 7.13 ± 0.7 6.7 ± 0.7 0.9

Pellet C Supernatant Microvilli ND – – 5.61 ± 0.96 0.6 ± 0.1 0.4 1.14 ± 0.15 0.7 ± 0.1 0.5 6.82 ± 0.77 9.7 ± 1.1 6.6 110 ± 6 19.3 ± 1.1 13.1

ND – – 8.08 ± 2.16 11.5 ± 3.1 0.6 ND – – 0.82 ± 0.2 15 ± 3.7 0.8 2.14 ± 0.41 4.9 ± 1.9 0.3

Pellet 2

MVs

Supernatant 3

2.08 ± 0.21 7 ± 0.7 1 7.23 ± 1.08 3.8 ± 0.6 0.5 4.04 ± 0.82 12.7 ± 2.6 1.8 2.13 ± 0.25 14.6 ± 1.7 2 14.06 ± 2.4 11.9 ± 2 1.7

ND – – 1.9 ± 0.6 0.3 ± 0.1 0.1 0.67 ± 0.02 0.7 ± 0.1 0.3 5.3 ± 0.85 11.8 ± 1.9 5.1 121 ± 13.3 33.4 ± 3.7 14.4

ND – – ND – – ND – – 0.46 ± 0.1 11.3 ± 2.4 0.5 1.22 ± 0.98 3.7 ± 3 0.1

Succinate dehydrogenase (marker of the inner mitochondrial membrane), NADH oxidase (both mitochondrial membranes and endoplasmic reticulum), acid phosphatase (lysosomes), leucine aminopeptidase (apical plasma membranes) and alkaline phosphatase (apical plasma membranes and matrix vesicles) activities were measured in all fractions obtained during the simultaneous isolation of microvilli and matrix vesicles from Saos-2 cell cultures. The enzyme activity quantifications are given in the Materials and methods section. Pellet A: cell debris, nuclei, mitochondria, lysosomes; Pellet B: basolateral membranes; Supernatant C: microsomal and cytosolic fractions. Pellet 2: cell debris, nuclei, mitochondria, lysosomes; Supernatant 3: digested extracellular matrix. Information about preparation of these fractions is provided in the Materials and methods section. (E: enrichment; ND: non-detected).

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Table 2 – Functional classification of proteins identified in Saos-2 cell microvilli and matrix vesicles. Category of proteins

Cytoskeletal proteins Cell adhesion proteins Extracellular matrix proteins Proteases Enzymes Transmembrane proteins Transporters/channels Calcium binding proteins Membrane trafficking proteins Signaling proteins Immune system proteins Chaperones Ubiquitin proteasome system proteins Protein synthesis Nucleic acid metabolism Others Total proteins

Microvilli

MVs

Common

N

(Previously identified)

N

(Previously identified)

N

42 17 1 12 82 13 30 12 61 50 8 25 23 42 8 25 451

(28) (6) (–) (3) (36) (3) (11) (8) (22) (18) (–) (16) (6) (20) (2) (3) (182)

33 14 6 7 43 11 20 9 39 32 4 16 14 20 4 10 282

(24) (10) (4) (3) (23) (5) (11) (7) (12) (13) (–) (5) (6) (6) (1) (4) (134)

33 14 1 7 42 9 18 9 35 28 4 16 14 20 4 8 262

The proteins identified in Saos-2 cell microvilli, matrix vesicles, and both (common) were classified into different functional categories according to the Human Protein Reference Database (http://www.hprd.org). The number of identified proteins (N) in each category and the number of proteins that had been previously identified in microvilli 38–41 or in MVs [10–12] are given.

cellular regions for translation [53]. In the microvillar proteomic analysis, we identified several proteins known to be involved in mRNA transport from the nucleus to a specific cell area: heterogeneous nuclear ribonucleoproteins, polyadenylate-binding protein 1 and tubulins [52,53] (Table 4). In addition, local translation implies the co-localization of the protein synthetic machinery with mRNAs. The presence of mRNA in MVs has already been described [54]. Many proteins that were detected in microvilli and MVs such as ribosomal proteins, aminoacyl-tRNA synthetases, and translation initiation and elongation factors are involved in protein synthesis (Table 4).

4.3.

Matrix vesicle biogenesis

Several observations indicate that MVs are formed by budding from the tip of mineralizing cell microvilli [2,3,55]. Microvilli contain a dense bundle of cross-linked actin microfilaments as a structural core [56]. The plus ends of actin filaments are located at the tip of microvilli, while the minus ends are anchored in the terminal web composed of a complex set of proteins including spectrin and myosins [56]. In addition to actin, several specific cytoskeletal components of apical microvilli were identified in MVs, namely ezrin, moesin, radixin, vinculin, plastin 1 and 3, actinin α1 and α4, Rac 1 and 3 and talin 1 (Supplemental Tables 1 and 2). These proteins, found in both MVs and microvilli, participate in the formation and maintenance of microvilli [57,58]. It was demonstrated that cytochalasin D, an inhibitor of actin microfilament assembly, stimulates MV release from microvilli of mineralizing cells [7,8]. Consistent with this observation, the actin core disruption is a necessary process for vesicle formation from kidney [59] and intestinal [60] microvilli. These findings are supported by our observations revealing the occurrence of actin-modulating proteins (cofilin

1, gelsolin and myristoylated alanine-rich C kinase substrate) in MVs and microvilli (Table 4). Cofilin 1 is a pH-sensitive actin-depolymerizing and severing protein [61]. Gelsolin is a calcium-regulated actin-modulating protein that prevents actin polymerization by end-blocking and severs the existing actin microfilaments when intracellular concentration of Ca2+ increases [62]. MARCKS is a protein crosslinking actin filaments and the plasma membrane. Its phosphorylation by protein kinase C or binding to Ca2+-calmodulin inhibits its activity and leads to actin cytoskeleton reorganization [63]. These three proteins could be involved in actin network retraction leading to MV budding from the apical microvilli and this process may be favored by high intracellular Ca2+ concentration. This is consistent with the massive Ca2+ release from mitochondria and the increase in Ca2+ concentration at sites of MV formation [6], and suggests that Ca2+ signaling is a crucial factor triggering MV formation. In addition to the longitudinal actin core, transverse crossbridges that connect the central core to the microvillar membrane and actin microfilaments to each other, seem to be implicated in the vesiculation of kidney microvilli. Rearrangement of the actin architecture is Ca2+- and ATPdependent in intestinal microvilli and probably involves contractile myosins [64]. Class I myosin IB and myosin IC as well as class II myosin heavy chain 9, myosin alkali light chain 6 and myosin regulatory light chain 3 were identified in both microvilli and MVs (Supplemental Table 1 and 2). Myosins belonging to different classes were identified as cytoskeletal components of intestinal microvilli and shown to function in microvillus contraction and vesiculation [65,66]. The occurrence of these myosins in microvilli and MVs suggests their role in MV release from the apical microvilli of Saos-2 cells. Myosin contraction requires ATP that may be supplied by glycolytic enzymes or brain creatine kinase (Supplemental Tables 1 and 2).

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Matrix vesicle functions

We also identified new proteins that could be involved in MV functions (Table 3). Among proteins that control Pi and PPi homeostasis, a lumenal inorganic pyrophosphatase 1 which hydrolyzes PPi was identified in MVs (Table 3). This protein may play a dual role in mineralization by providing Pi and hydrolyzing PPi, an inhibitor of mineral growth [17,18]. Furthermore, sphingomyelin phosphodiesterase 3 was found in MVs (Table 3). This is a transmembrane enzyme, with an intravesicular active site, catalyzing the hydrolysis of sphin-

gomyelin to generate ceramide and phosphocholine [67]. Phosphocholine, as a substrate of the luminal PHOSPHO1 (Table 3), could be further hydrolyzed to generate Pi [21]. Therefore, sphingomyelin phosphodiesterase 3 may contribute to mineralization by indirectly increasing Pi level. A mutation of its gene leads to osteogenesis imperfecta in mice [68]. It was proposed that Pi transport from the ECM to the MV lumen may be performed by sodium-dependent Pi transporter (a member of the type III Glvr-1gene family) [22] or by a not strictly sodium-dependent alkaline pH-specific Pi transporter [23]. Their presence in MVs was suggested on the

Table 3 – List of selected proteins involved in matrix vesicle functions. Accession number

MW (Da)

Score

N

SC (%)

57305 112896 133930 40329 71081 32660 29713 99930

2723 990 366 188 172 150 137 134

22 26 11 3 5 4 2 3

66.8 35.8 13.7 14.0 11.0 24.2 7.9 5.0

Hydrolysis of PPi [15,17,18] Hydrolysis of ATP [20] Hydrolysis of ATP [10] Hydrolysis of ATP [11] Mineralization Hydrolysis of PPi Provides Pi [21] Provides PPi [16]

75873 40411 35937 38714 36085 54390 52739 21676

873 1627 710 593 417 358 249 197

23 13 13 9 9 7 6 4

49.9 55.2 59.7 39.6 36.4 18.8 15.6 24.2

Ca2+-influx into MVs [25] Ca2+-influx into MVs [25] Mineral nucleator [28] Ca2+-influx into MVs [10–12] Ca2+–influx into MVs [10–12] Ca2+-influx into MVs [11,12] Ca2+-influx into MVs [10] Mineral nucleator

Regulation of MV ionic balance, volume and pH 19913432 Vacuolar H+ ATPase subunit D 134288865 SLC4A7 (sodium bicarbonate cotransporter) 119582796 SLC12A2 (Na+/K+/Cl- transporter)

40329 136044 131447

188 140 126

3 3 3

14.0 3.1 3.8

Regulation of pH Regulation of pH Regulation of MV volume

MV-ECM adhesion, mineral growth and localization 4503053 Hyaluronan and proteoglycan link protein 1 13477169 Vitronectin 119616317 Chondroitin sulfate proteoglycan 2 19743813 Integrin β1 4504747 Integrin α3 31657142 Integrin α1 62089374 Integrin αV 119631391 Integrin α4 20127446 Integrin β5 56237029 Integrin α5 4507877 Vinculin 16753233 Talin 1 29789006 Kindlin 2

40165 54305 372819 91620 118755 130847 116038 114899 88054 114536 123799 269667 77860

484 86 62 489 462 401 390 354 239 147 509 406 97

9 2 2 16 11 8 13 11 7 4 11 11 2

39.6 5.9 4.7 28.5 13.7 8.7 15.8 16.4 13.3 5.1 12.8 6.1 4.1

MV-ECM adhesion [11,12] MV-ECM adhesion [12] MV-ECM adhesion [10–12] MV-ECM adhesion [10,11] MV-ECM adhesion [11] MV-ECM adhesion MV-ECM adhesion [10,11] MV-ECM adhesion MV-ECM adhesion [11] MV-ECM adhesion MV adhesion/motility [10] MV adhesion/motility MV adhesion/motility

42200 26224

136 135

7 2

25.7 11.1

Stimulates ECM degradation Inhibits ECM deposition

71081

172

5

11.0

MV membrane lysis

Pi and PPi homeostasis 116734717 21361181 14286105 19913432 89954531 11056044 30425420 119568427

Protein name Tissue non-specific alkaline phosphatase Na+/K+ ATPase α1 Plasma membrane Ca2+ ATPase type 4 Vacuolar H+ ATPase subunit D Sphingomyelin phosphodiesterase 3 Inorganic pyrophosphatase 1 PHOSPHO1 NPP1

Ca2+ ion homeostasis and mineral nucleation 71773329 Annexin A6 18645167 Annexin A2 809185 Annexin A5 4502101 Annexin A1 1703319 Annexin A4 1703322 Annexin A11 55584155 Annexin A7 4507207 Sorcin

ECM remodeling 20146101 19923989

Basigin Collagen triple helix repeat-containing 1

MV membrane breakdown and mineral release 89954531 Sphingomyelin phosphodiesterase 3

Function

List of selected proteins with their known or proposed role in MV functions. The name, accession number in the NCBI protein database, molecular weight (MW), highest identification score, number of peptide matches (N), sequence coverage (SC) and functions of each detected protein are provided.

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Table 4 – List of selected proteins which may be involved in matrix vesicle biogenesis from Saos-2 cell microvilli. Accession number

Protein name

Microvilli

MVs

Function

Score N SC (%) Score N SC (%)

Lipid and protein targeting to MVs 4557803 Niemann Pick C1 protein 4503015 Copine III 73536235 Rab14 119590297 Rab4A 49168476 Rab11B 7657675 Vesicle-associated membrane protein 2 5032137 Vesicle-associated membrane protein 7 18645167 Annexin A2 3929617 SNAP α

242 223 220 139 426 221 171 803 113

4 3 3 2 4 2 2 14 3

4.2 7.1 27.1 13.2 25.2 25.9 15.5 61.4 12.2

81 124 168 – 325 – – 753 159

2 2 3 – 4 – – 13 5

2.3 4.7 24.9 – 24.3 – – 55.2 19.7

Intracellular trafficking of cholesterol Membrane trafficking Apical membrane targeting [47] Apical membrane targeting [49] Apical membrane targeting [49] Apical membrane fusion [50] Apical membrane fusion [50] Apical membrane fusion [51] Apical membrane fusion [51]

Soluble protein targeting to MVs 460789 Heterogeneous nuclear ribonucleoprotein K 119612222 Polyadenylate-binding protein 1 57209813 Tubulin β 15718687 Ribosomal protein S3 4503483 Eukaryotic translation elongation factor 2

79 214 861 287 222

3 4 19 8 3

10.4 13.1 73.9 43.6 5.0

– – 820 725 201

– – 17 9 4

– – 60.6 47.3 6.1

mRNA nucleocytoplasmic transport [52,53] mRNA nucleocytoplasmic transport [52,53] mRNA nucleocytoplasmic transport [52,53] On-site protein synthesis [54] On-site protein synthesis [54]

860 1067 498 452 161 354 2730 609 312 258 653 589 302

16 10 7 6 3 4 44 9 7 3 9 14 5

9.3 44.0 15.1 40.1 19.7 35.5 32.3 11.9 14.0 29.7 37.2 36.9 23.1

– 765 297 372 103 289 2775 207 111 – 568 454 296

– 10 10 8 2 2 39 5 4 – 5 11 4

– 44.0 24.4 48.2 14.5 25.3 29.5 6.8 8.9 – 20.4 31.1 18.6

Actin microfilament movement Shape and motility Actin anchorage to plasma membrane [57] Actin anchorage to plasma membrane [63] Ca2+-regulated actin severing protein [62] pH-sensitive actin severing protein [61] Actin cytoskeleton contraction [64] Actin cytoskeleton contraction [65] Ca2+-dependent regulation of actin [63] Ca2+-dependent regulation of actin [63] Glycolytic enzyme providing ATP Glycolytic enzyme providing ATP Enzyme providing ATP

MV budding 119608217 4501885 4505257 187387 55960300 5031635 12667788 44889481 62088530 825635 4505763 35505 180570

and release from apical microvilli Spectrin α, non-erythrocytic 1 (α-fodrin) β-actin Moesin MARCKS Gelsolin Cofilin 1 Myosin heavy polypeptide 9 non-muscle Myosin IB Protein kinase Cα Calmodulin 1 Phosphoglycerate kinase 1 Pyruvate kinase 3 Creatine kinase brain type

List of selected proteins with their proposed role in MV biogenesis. The name, accession number for the NCBI protein database, molecular weight (MW), highest identification score, number of peptide matches (N), sequence coverage (SC) and functions of each detected proteins are provided.

basis of transport activity. However, they could not be detected by proteomic analyses [10, 11 and present work]. A Pi transporter may not exist in MVs since a recent report proposed that skeletal calcification requires intravesicular Pi production by PHOSPHO1 during the initiation of mineralization while TNAP, NPP1, collagen and other ECM proteins are necessary for extravesicular progression of mineralization [69]. The intravesicular initiation of mineralization also requires Ca2+ influx into MVs and mineral nucleators. A voltagedependent Ca2+ channel may be a new complementary candidate for a transport system controling Ca2+ homeostasis in MVs (Supplemental Table 2). Sorcin (Table 3) which exhibits the ability to translocate from the cytosol to the membrane upon Ca2+-binding may play a role in nucleation [70]. HA formation leads to the release of H+. However, acidic pH inhibits mineral deposition and therefore pH must be regulated. We identified a protein which could adjust intravesicular pH, namely SLC4A7, a bicarbonate transporter (Table 3), whose presence has been previously speculated [29]. Moreover, the vacuolar proton pump ATPase may also regulate the intravesicular pH (Table 3). HA formation requires H2O molecules in addition to Ca2+ ions and Pi. SLC12A2, a Na+/K+/

Cl− co-transporter, another protein identified in MVs, may be involved in the regulation of intravesicular ionic balance and volume [71] (Table 3). A crucial step between the intravesicular initiation of mineralization and the extravesicular propagation of mineralization is the release of the mineral from MVs. Again, phospholipases A2 [30] and D [31], both detected in MVs, were suggested to be involved in MV membrane maintenance or breakdown. The sphingomyelin phosphodiesterase 3 (Table 3) may regulate lipid composition of the MV membrane and favor MV membrane breakdown leading to mineral release [30,68]. Furthermore, the interactions between the extracellular matrix and MVs are required to control mineral growth and localization. We identified several integrins that form receptors for extracellular matrix components (Table 3). Kindlin 2, talin 1 and vinculin (Table 3) which connect ECM components to the actin network may support MV adhesion and motility. In addition, MV preparations contained basigin, a matrix metalloprotease stimulator, and collagen triple helix repeat-containing protein 1 (Table 3), a negative regulator of collagen matrix deposition. Both these proteins may contribute to matrix degradation and facilitate the access of MVs to areas where mineralization takes place [33]. Degradation of

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the extracellular matrix also leads to the release of the transforming growth factor family members which play a role in paracrine/autocrine regulation of osseous cells [34].

5.

Conclusion

In this report, we characterized the proteome of MVs released from human mineralizing osteoblast-like Saos-2 cells and compared it to those described for MVs originating from various cell types and species. We succeeded in identifying novel proteins that may regulate PPi and Pi homeostasis (inorganic pyrophosphatase 1), Ca2+ ion homeostasis (voltagedependent Ca2+ channel and sorcin), intravesicular pH (vacuolar H+-ATPase and SLC4A7, a sodium bicarbonate cotransporter) or lipid composition of MV membrane (sphingomyelin phosphodiesterase 3). Our report also provided the first proteome of apical microvilli isolated from osteoblast-like cells. The comparison of the MV proteome with that of microvilli showed 93% of homology in protein composition. The occurrence of vesicular trafficking molecules in both microvilli and MVs pointed up to the apical trafficking as the mechanism responsible for MV membrane lipid and protein enrichment. Soluble proteins are targeted to the MV lumen through on-site translation as demonstrated by the presence of the protein synthetic machinery in the apical microvilli. Finally, the presence of several cytoskeletal (gelsolin and cofilin 1) and contractile motor proteins (class I, II and VI myosins) provided new insights into the mechanism of MV budding and pinching off the apical microvilli and of their release into the ECM. Supplementary materials related to this article can be foundonline at doi:10.1016/j.jprot.2011.04.005.

Acknowledgements This work was supported by a grant N N401 140639 from the Polish Ministry of Science and Higher Education, a FrenchPolish cooperation grant PICS N° 5096 and by the Rhône-Alpes region.

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