Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

Mineralization Profile of Annexin A6-Harbouring Proteoliposomes: Shedding Light on the Role of Annexin A6 on Matrix Vesicle-Mediated Mineralization.

The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles' nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS-CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS-CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles' nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS-CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.

Annexins A2, A6 and Fetuin-A Affect the Process of Mineralization in Vesicles Derived from Human Osteoblastic hFOB 1.19 and Osteosarcoma Saos-2 Cells.

The mineralization process is initiated by osteoblasts and chondrocytes during intramembranous and endochondral ossifications, respectively. Both types of cells release matrix vesicles (MVs), which accumulate Pi and Ca2+ and form apatites in their lumen. Tissue non-specific alkaline phosphatase (TNAP), a mineralization marker, is highly enriched in MVs, in which it removes inorganic pyrophosphate (PPi), an inhibitor of apatite formation. MVs then bud from the microvilli of mature osteoblasts or hypertrophic chondrocytes and, thanks to the action of the acto-myosin cortex, become released to the extracellular matrix (ECM), where they bind to collagen fibers and propagate mineral growth. In this report, we compared the mineralization ability of human fetal osteoblastic cell line (hFOB 1.19 cells) with that of osteosarcoma cell line (Saos-2 cells). Both types of cells were able to mineralize in an osteogenic medium containing ascorbic acid and beta glycerophosphate. The composition of calcium and phosphate compounds in cytoplasmic vesicles was distinct from that in extracellular vesicles (mostly MVs) released after collagenase-digestion. Apatites were identified only in MVs derived from Saos-2 cells, while MVs from hFOB 1.19 cells contained amorphous calcium phosphate complexes. In addition, AnxA6 and AnxA2 (nucleators of mineralization) increased mineralization in the sub-membrane region in strongly mineralizing Saos-2 osteosarcoma, where they co-localized with TNAP, whereas in less mineralizing hFOB 1.19 osteoblasts, AnxA6, and AnxA2 co-localizations with TNAP were less visible in the membrane. We also observed a reduction in the level of fetuin-A (FetuA), an inhibitor of mineralization in ECM, following treatment with TNAP and Ca channels inhibitors, especially in osteosarcoma cells. Moreover, a fraction of FetuA was translocated from the cytoplasm towards the plasma membrane during the stimulation of Saos-2 cells, while this displacement was less pronounced in stimulated hFOB 19 cells. In summary, osteosarcoma Saos-2 cells had a better ability to mineralize than osteoblastic hFOB 1.19 cells. The formation of apatites was observed in Saos-2 cells, while only complexes of calcium and phosphate were identified in hFOB 1.19 cells. This was also evidenced by a more pronounced accumulation of AnxA2, AnxA6, FetuA in the plasma membrane, where they were partly co-localized with TNAP in Saos-2 cells, in comparison to hFOB 1.19 cells. This suggests that both activators (AnxA2, AnxA6) and inhibitors (FetuA) of mineralization were recruited to the membrane and co-localized with TNAP to take part in the process of mineralization.

Localization of Annexin A6 in Matrix Vesicles During Physiological Mineralization.

Annexin A6 (AnxA6) is the largest member of the annexin family of proteins present in matrix vesicles (MVs). MVs are a special class of extracellular vesicles that serve as a nucleation site during cartilage, bone, and mantle dentin mineralization. In this study, we assessed the localization of AnxA6 in the MV membrane bilayer using native MVs and MV biomimetics. Biochemical analyses revealed that AnxA6 in MVs can be divided into three distinct groups. The first group corresponds to Ca2+-bound AnxA6 interacting with the inner leaflet of the MV membrane. The second group corresponds to AnxA6 localized on the surface of the outer leaflet. The third group corresponds to AnxA6 inserted in the membrane's hydrophobic bilayer and co-localized with cholesterol (Chol). Using monolayers and proteoliposomes composed of either dipalmitoylphosphatidylcholine (DPPC) to mimic the outer leaflet of the MV membrane bilayer or a 9:1 DPPC:dipalmitoylphosphatidylserine (DPPS) mixture to mimic the inner leaflet, with and without Ca2+, we confirmed that, in agreement with the biochemical data, AnxA6 interacted differently with the MV membrane. Thermodynamic analyses based on the measurement of surface pressure exclusion (πexc), enthalpy (ΔH), and phase transition cooperativity (Δt1/2) showed that AnxA6 interacted with DPPC and 9:1 DPPC:DPPS systems and that this interaction increased in the presence of Chol. The selective recruitment of AnxA6 by Chol was observed in MVs as probed by the addition of methyl-ß-cyclodextrin (MßCD). AnxA6-lipid interaction was also Ca2+-dependent, as evidenced by the increase in πexc in negatively charged 9:1 DPPC:DPPS monolayers and the decrease in ΔH in 9:1 DPPC:DPPS proteoliposomes caused by the addition of AnxA6 in the presence of Ca2+ compared to DPPC zwitterionic bilayers. The interaction of AnxA6 with DPPC and 9:1 DPPC:DPPS systems was distinct even in the absence of Ca2+ as observed by the larger change in Δt1/2 in 9:1 DPPC:DPPS vesicles as compared to DPPC vesicles. Protrusions on the surface of DPPC proteoliposomes observed by atomic force microscopy suggested that oligomeric AnxA6 interacted with the vesicle membrane. Further work is needed to delineate possible functions of AnxA6 at its different localizations and ways of interaction with lipids.

Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization.

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (-Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

Src and ROCK Kinases Differentially Regulate Mineralization of Human Osteosarcoma Saos-2 Cells.

Osteoblasts initiate bone mineralization by releasing matrix vesicles (MVs) into the extracellular matrix (ECM). MVs promote the nucleation process of apatite formation from Ca2+ and Pi in their lumen and bud from the microvilli of osteoblasts during bone development. Tissue non-specific alkaline phosphatase (TNAP) as well as annexins (among them, AnxA6) are abundant proteins in MVs that are engaged in mineralization. In addition, sarcoma proto-oncogene tyrosine-protein (Src) kinase and Rho-associated coiled-coil (ROCK) kinases, which are involved in vesicular transport, may also regulate the mineralization process. Upon stimulation in osteogenic medium containing 50 µg/mL of ascorbic acid (AA) and 7.5 mM of ß-glycerophosphate (ß-GP), human osteosarcoma Saos-2 cells initiated mineralization, as evidenced by Alizarin Red-S (AR-S) staining, TNAP activity, and the partial translocation of AnxA6 from cytoplasm to the plasma membrane. The addition of 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d] pyrimidine (PP2), which is an inhibitor of Src kinase, significantly inhibited the mineralization process when evaluated by the above criteria. In contrast, the addition of (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide hydrochloride (Y-27632), which is an inhibitor of ROCK kinase, did not affect significantly the mineralization induced in stimulated Saos-2 cells as denoted by AR-S and TNAP activity. In conclusion, mineralization by human osteosarcoma Saos-2 cells seems to be differently regulated by Src and ROCK kinases.

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models.

BACKGROUND: Matrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100-300nm diameter harboring the biochemical machinery needed to induce mineralization. SCOPE OF THE REVIEW: The review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs. MAJOR CONCLUSIONS: MVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies. GENERAL SIGNIFICANCE: MVs have been extensively investigated owing to their roles in skeletal and ectopic mineralization. MVs serve as a model system for lipid raft structures, and for the mechanisms of genesis and release of extracellular vesicles.

Vesicles of the intracellular and extracellular transport - key structures in the process of tissue differentiation towards bone and cartilage.

Differentiation of cells of the skeletal tissue, such as osteoblasts and chondrocytes, into mineralization-competent cells is a necessary step of the physiological process of bone and cartilage mineralization. Vascular cell calcification accompanies a pathological process of atherosclerotic plaque formation, which occurs due to trans-differentiation of vascular smooth muscle cells into cells resembling bone mineralization-competent cells. The activity of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme necessary for physiological mineralization, is also induced in vascular cells in response to inflammation. TNAP acquires its mineralizing function when anchored to the plasma membrane (PM) of mineralizing cells and to the surface of vesicles derived from these cells. Numerous important reports indicate that various types of vesicles play a crucial role in initiating cell differentiation. In this review, we would like to highlight various functions of different types of vesicular structures of the cellular transport machinery such as intracellular vesicles (IVs), extracellular vesicles (EVs) or matrix vesicles (MVs) at distinct stages of both physiological and pathological processes of tissue differentiation.

Functions of Rho family of small GTPases and Rho-associated coiled-coil kinases in bone cells during differentiation and mineralization.

BACKGROUND: Members of Rho-associated coiled-coil kinases (ROCKs) are effectors of Rho family of small GTPases. ROCKs have multiple functions that include regulation of cellular contraction and polarity, adhesion, motility, proliferation, apoptosis, differentiation, maturation and remodeling of the extracellular matrix (ECM). SCOPE OF THE REVIEW: Here, we focus on the action of RhoA and RhoA effectors, ROCK1 and ROCK2, in cells related to tissue mineralization: mesenchymal stem cells, chondrocytes, preosteoblasts, osteoblasts, osteocytes, lining cells and osteoclasts. MAJOR CONCLUSIONS: The activation of the RhoA/ROCK pathway promotes stress fiber formation and reduces chondrocyte and osteogenic differentiations, in contrast to that in mesenchymal stem cells which stimulated the osteogenic and the chondrogenic differentiation. The effects of Rac1 and Cdc42 in promoting chondrocyte hypertrophy and of Rac1, Rac2 and Cdc42 in osteoclast are discussed. In addition, members of the Rho family of GTPases such Rac1, Rac2, Rac3 and Cdc42, acting upstream of ROCK and/or other protein effectors, may compensate the actions of RhoA, affecting directly or indirectly the actions of ROCKs as well as other protein effectors. GENERAL SIGNIFICANCE: ROCK activity can trigger cartilage degradation and affect bone formation, therefore these kinases may represent a possible therapeutic target to treat osteoarthritis and osseous diseases. Inhibition of Rho/ROCK activity in chondrocytes prevents cartilage degradation, stimulate mineralization of osteoblasts and facilitate bone formation around implanted metals. Treatment with osteoprotegerin results in a significant decrease in the expression of Rho GTPases, ROCK1 and ROCK2, reducing bone resorption. Inhibition of ROCK signaling increases osteoblast differentiation in a topography-dependent manner.

Cross-talk between Src kinases and Rho small GTPases regulates biomineralization and simplify imaging of the mineralization process.

The process of physiological mineralization that occurs during bone ossification is a tightly regulated cascade of molecular events leading to formation of bony skeleton. Its deregulation associated with aging leads to pathological mineralization not only in osseous but also in soft tissues. Recent discoveries in the field of bone biology indicate the participation of the Src family of tyrosine kinases as well as the Rho family of small GTPases in mineral formation. Cross-talk between these two signaling pathways is activated during an early step of the mineralization process, and leads to reorganization of the cytoskeleton of mineralization-competent cells and to matrix vesicles release. The understanding of mechanisms of the cross-talk between Src kinases and Rho small GTPases that regulate the mineralization process is crucial for the development of novel simply imaging techniques and therapeutic strategies in relation to pathological mineralization.

Two-Step Membrane Binding of NDPK-B Induces Membrane Fluidity Decrease and Changes in Lipid Lateral Organization and Protein Cluster Formation.

Nucleoside diphosphate kinases (NDPKs) are crucial elements in a wide array of cellular physiological or pathophysiological processes such as apoptosis, proliferation, or metastasis formation. Among the NDPK isoenzymes, NDPK-B, a cytoplasmic protein, was reported to be associated with several biological membranes such as plasma or endoplasmic reticulum membranes. Using several membrane models (liposomes, lipid monolayers, and supported lipid bilayers) associated with biophysical approaches, we show that lipid membrane binding occurs in a two-step process: first, initiation by a strong electrostatic adsorption process and followed by shallow penetration of the protein within the membrane. The NDPK-B binding leads to a decrease in membrane fluidity and formation of protein patches. The ability of NDPK-B to form microdomains at the membrane level may be related to protein-protein interactions triggered by its association with anionic phospholipids. Such accumulation of NDPK-B would amplify its effects in functional platform formation and protein recruitment at the membrane.

Membranes and pathophysiological mineralization.

Vascular calcification accompanies the pathological process of atherosclerotic plaque formation. Artery calcification results from trans-differentiation of vascular smooth muscle cells (VSMCs) into cells resembling mineralization-competent cells such as osteoblasts and chondrocytes. The activity of tissue-nonspecific alkaline phosphatase (TNAP), a GPI-anchored enzyme necessary for physiological mineralization, is induced in VSMCs in response to inflammation. TNAP achieves its mineralizing function being anchored to plasma membrane of mineralizing cells and to the surface of their derived matrix vesicles (MVs), and numerous important reports indicate that membranes play a crucial role in initiating the crystal formation. In this review, we would like to highlight various functions of lipids and proteins associated to membranes at different stages of both physiological mineralization and vascular calcification, with an emphasis on the pathological process of atherosclerotic plaque formation.

Exploring NMR methods as a tool to select suitable fluorescent nucleotide analogues.

Fluorescent analogues provide important tools for biochemical/biophysical research. However, the analogues contain chemical modifications much larger than those known to affect ligand-binding, such as the inversion of a carbon centre or substitution of an atom. We lack experimental tools and protocols to select the most appropriate fluorescent analogue. Herein, we use several NMR spectroscopy methods, including Saturation Transfer Difference (STD), STD competition and transferred nuclear Overhauser effect spectroscopy (Tr-NOESY), as tools to select appropriate fluorescent probes. Annexin A6 (AnxA6) is a ubiquitous protein that forms in vitro GTP-induced ion channels. We used this protein as a model and screened guanosine triphosphate (GTP) and four fluorescent analogues against AnxA6. STD reported that the GTP moiety of all ligands made similar contacts with the protein, despite additional interactions between the fluorescent tags and AnxA6. Competition STD experiments verified that the analogues and GTP bind to the same site. Tr-NOESY indicated that the bound conformation of the base relative to ribose is altered for some analogues compared to GTP. MANT-GTP or the BODIPY thioester of guanosine 5'-O-(3-thiotriphosphate) are the most suitable fluorescent analogues for AnxA6, according to NMR. These results reveal NMR as a useful technique to select and design proper fluorescent tags for biochemical/biophysical assays.

Do Media Extracellular Vesicles and Extracellular Vesicles Bound to the Extracellular Matrix Represent Distinct Types of Vesicles?

Mineralization-competent cells, including hypertrophic chondrocytes, mature osteoblasts, and osteogenic-differentiated smooth muscle cells secrete media extracellular vesicles (media vesicles) and extracellular vesicles bound to the extracellular matrix (matrix vesicles). Media vesicles are purified directly from the extracellular medium. On the other hand, matrix vesicles are purified after discarding the extracellular medium and subjecting the cells embedded in the extracellular matrix or bone or cartilage tissues to an enzymatic treatment. Several pieces of experimental evidence indicated that matrix vesicles and media vesicles isolated from the same types of mineralizing cells have distinct lipid and protein composition as well as functions. These findings support the view that matrix vesicles and media vesicles released by mineralizing cells have different functions in mineralized tissues due to their location, which is anchored to the extracellular matrix versus free-floating.

TNAP as a therapeutic target for cardiovascular calcification: a discussion of its pleiotropic functions in the body.

Cardiovascular calcification (CVC) is associated with increased morbidity and mortality. It develops in several diseases and locations, such as in the tunica intima in atherosclerosis plaques, in the tunica media in type 2 diabetes and chronic kidney disease, and in aortic valves. In spite of the wide occurrence of CVC and its detrimental effects on cardiovascular diseases (CVD), no treatment is yet available. Most of CVC involve mechanisms similar to those occurring during endochondral and/or intramembranous ossification. Logically, since tissue-nonspecific alkaline phosphatase (TNAP) is the key-enzyme responsible for skeletal/dental mineralization, it is a promising target to limit CVC. Tools have recently been developed to inhibit its activity and preclinical studies conducted in animal models of vascular calcification already provided promising results. Nevertheless, as its name indicates, TNAP is ubiquitous and recent data indicate that it dephosphorylates different substrates in vivo to participate in other important physiological functions besides mineralization. For instance, TNAP is involved in the metabolism of pyridoxal phosphate and the production of neurotransmitters. TNAP has also been described as an anti-inflammatory enzyme able to dephosphorylate adenosine nucleotides and lipopolysaccharide. A better understanding of the full spectrum of TNAP's functions is needed to better characterize the effects of TNAP inhibition in diseases associated with CVC. In this review, after a brief description of the different types of CVC, we describe the newly uncovered additional functions of TNAP and discuss the expected consequences of its systemic inhibition in vivo.

Fluorescence evidence of annexin A6 translocation across membrane in model matrix vesicles during apatite formation.

Matrix vesicles (MVs) are 100-300 nm spherical structures released by mineralization competent cells to initiate formation of apatite, the mineral component in bones. Among proteins present in MVs, annexin A6 (AnxA6) is thought to be ubiquitously distributed in the MVs' lumen, on the surface of the internal and external leaflets of the membrane and also inserted in the lipid bilayer. To determine the molecular mechanism(s) that lead to the different locations of AnxA6, we hypothesized the occurrence of a pH drop during the mineralization. Such a change would induce the AnxA6 protonation, which in turn, and because of its isoelectric point of 5.41, would change the protein hydrophobicity facilitating its insertion into the MVs' bilayer. The various distributions of AnxA6 are likely to disturb membrane phospholipid organization. To examine this possibility, we used fluorescein as pH reporter, and established that pH decreased inside MVs during apatite formation. Then, 4-(14-phenyldibenzo[a,c]phenazin-9(14H)-yl)-phenol, a vibration-induced emission fluorescent probe, was used as a reporter of changes in membrane organization occurring with the varying mode of AnxA6 binding. Proteoliposomes containing AnxA6 and 1,2-Dimyristoyl-sn-glycero-3phosphocholine (DMPC) or 1,2-Dimyristoyl-sn-glycero-3phosphocholine: 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (DMPC:DPPS 9:1), to mimic the external and internal MV membrane leaflet, respectively, served as biomimetic models to investigate the nature of AnxA6 binding. Addition of Anx6 to DMPC at pH 7.4 and 5.4, or DMPC:DPPS (9:1) at pH 7.4 induced a decrease in membrane fluidity, consistent with AnxA6 interactions with the bilayer surface. In contrast, AnxA6 addition to DMPC:DPPS (9:1) at pH 5.4 increased the fluidity of the membrane. This latest result was interpreted as reflecting the insertion of AnxA6 into the bilayer. Taken together, these findings point to a possible mechanism of AnxA6 translocation in MVs from the surface of the internal leaflet into the phospholipid bilayer stimulated upon acidification of the MVs' lumen during formation of apatite.

Matrix vesicles isolated from mineralization-competent Saos-2 cells are selectively enriched with annexins and S100 proteins.

Matrix vesicles (MVs) are cell-derived membranous entities crucial for mineral formation in the extracellular matrix. One of the dominant groups of constitutive proteins present in MVs, recognised as regulators of mineralization in norm and pathology, are annexins. In this report, besides the annexins already described (AnxA2 and AnxA6), we identified AnxA1 and AnxA7, but not AnxA4, to become selectively enriched in MVs of Saos-2 cells upon stimulation for mineralization. Among them, AnxA6 was found to be almost EGTA-non extractable from matrix vesicles. Moreover, our report provides the first evidence of annexin-binding S100 proteins to be present in MVs of mineralizing cells. We observed that S100A10 and S100A6, but not S100A11, were selectively translocated to the MVs of Saos-2 cells upon mineralization. This observation provides the rationale for more detailed studies on the role of annexin-S100 interactions in MV-mediated mineralization.

Characterization of caged compounds binding to proteins by NMR spectroscopy.

Photolysable caged ligands are used to investigate protein function and activity. Here, we investigate the binding properties of caged nucleotides and their photo released products to well established but evolutionary and structurally unrelated nucleotide-binding proteins, rabbit muscle creatine kinase (RMCK) and human annexin A6 (hAnxA6), using saturation transfer difference NMR spectroscopy. We detect the binding of the caged nucleotides and discuss the general implications on interpreting data collected with photolysable caged ligands using different techniques. Strategies to avoid non-specific binding of caged compound to certain proteins are also suggested.

Cholesterol as a factor regulating intracellular localization of annexin A6 in Niemann-Pick type C human skin fibroblasts.

Lysosome-like storage organelles (LSOs) play a crucial role in excessive accumulation of cholesterol in the Niemann-Pick type C (NPC) disease characterized by altered vesicular traffic of lipids. Annexin A6 (AnxA6) is mainly present in cytosol but upon elevation of [Ca(2+)](in) binds to membranes. In addition, a pH or cholesterol-dependent mechanism of AnxA6 interaction with membranes was described. We found a several fold enrichment of AnxA6 in LSO compartment in fibroblasts isolated from NPC patients in comparison with fibroblasts from healthy individuals. We observed that AnxA6 relocates from cytosol to LSOs in a cholesterol-dependent manner. Cholesterol depletion caused reduction in the binding of AnxA6. Moreover, we found that in NPC cells AnxA6 translocates to the perinuclear region containing late endosomes (LE) loaded with cholesterol. We conclude that AnxA6 may participate in formation of cholesterol-rich platforms on LE and therefore may contribute to the pathology of the NPC disease.

Matrix vesicles originate from apical membrane microvilli of mineralizing osteoblast-like Saos-2 cells.

In bone, mineralization is tightly regulated by osteoblasts and hypertrophic chondrocytes which release matrix vesicles (MVs) and control extracellular ionic conditions and matrix composition. MVs are the initial sites of hydroxyapatite (HA) mineral formation. Despite growing knowledge about their morphology and function, their biogenesis is not well understood. The purpose of this work was to determine the source of MVs in osteoblast lineage, Saos-2 cells, and to check whether MVs originated from microvilli. Microvilli were isolated from the apical plasma membrane of Saos-2 cells. Their morphology, structure, and function were compared with those of MVs. The role of actin network in MV release was investigated by using microfilament perturbing drugs. When examined by electron microscopy MVs and microvillar vesicles were found to exhibit similar morphology with trilaminar membranes and diameters in the same range. Both types of vesicles were able to induce HA formation. Their electrophoretic profiles displayed analogous enrichment in alkaline phosphatase, Na(+)/K(+) ATPase, and annexins A2 and A6. MVs and microvillar vesicles exhibited almost the same lipid composition with a higher content of cholesterol, sphingomyelin, and phosphatidylserine as compared to plasma membrane. Finally, cytochalasin D, which inhibits actin polymerization, was found to stimulate release of MVs. Our findings were consistent with the hypothesis that MVs originated from cell microvilli and that actin filament disassembly was involved in their biogenesis.