Agnieszka Strzelecka-Kiliszek

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. J. Cell. Biochem. 106: 127–138, 2009.

Lyn and Syk Kinases Are Sequentially Engaged in Phagocytosis Mediated by FcγR

Recent data indicate that phagocytosis mediated by FcγRs is controlled by the Src and Syk families of protein tyrosine kinases. In this study, we demonstrate a sequential involvement of Lyn and Syk in the phagocytosis of IgG-coated particles. The particles isolated at the stage of their binding to FcγRs (4°C) were accompanied by high amounts of Lyn, in addition to the signaling γ-chain of FcγRs. Simultaneously, the particle binding induced rapid tyrosine phosphorylation of numerous proteins. During synchronized internalization of the particles induced by shifting the cell to 37°C, Syk kinase and Src homology 2-containing tyrosine phosphatase-1 (SHP-1) were associated with the formed phagosomes. At this step, most of the proteins were dephosphorylated, although some underwent further tyrosine phosphorylation. Quantitative immunoelectron microscopy studies confirmed that Lyn accumulated under the plasma membrane beneath the bound particles. High amounts of the γ-chain and tyrosine-phosphorylated proteins were also observed under the bound particles. When the particles were internalized, the γ-chain was still detected in the region of the phagosomes, while amounts of Lyn were markedly reduced. In contrast, the vicinity of the phagosomes was heavily decorated with anti-Syk and anti-SHP-1 Abs. The local level of protein tyrosine phosphorylation was reduced. The data indicate that the accumulation of Lyn during the binding of IgG-coated particles to FcγRs correlated with strong tyrosine phosphorylation of numerous proteins, suggesting an initiating role for Lyn in protein phosphorylation at the onset of the phagocytosis. Syk kinase and SHP-1 phosphatase are mainly engaged at the stage of particle internalization.

Binding of IgG-Opsonized Particles to FcγR Is an Active Stage of Phagocytosis That Involves Receptor Clustering and Phosphorylation

FcγR mediate the phagocytosis of IgG-coated particles and the clearance of IgG immune complexes. By dissecting binding from internalization of the particles, we found that the binding stage, rather than particle internalization, triggered tyrosine phosphorylation of FcγR and accompanying proteins. High amounts of Lyn kinase were found to associate with particles isolated at the binding stage from J774 cells. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an Src kinase inhibitor, but not piceatannol, an inhibitor of Syk kinase, reduced the amount of Lyn associated with the bound particles and simultaneously diminished the binding of IgG-coated particles. Studies of baby hamster kidney cells transfected with wild-type and mutant FcγRIIA revealed that the ability of the receptor to bind particles was significantly reduced when phosphorylation of the receptor was abrogated by Y298F substitution in the receptor signaling motif. Under these conditions, binding of immune complexes of aggregated IgG was depressed to a lesser extent. A similar effect was exerted on the binding ability of wild-type FcγRIIA by PP2. Moreover, expression of mutant kinase-inactive Lyn K275R inhibited both FcγRIIA phosphorylation and IgG-opsonized particle binding. To gain insight into the mechanism by which protein tyrosine phosphorylation can control FcγR-mediated binding, we investigated the efficiency of clustering of wild-type and Y298F-substituted FcγRIIA upon binding of immune complexes. We found that a lack of FcγRIIA phosphorylation led to an impairment of receptor clustering. The results indicate that phosphorylation of FcγR and accompanying proteins, dependent on Src kinase activity, facilitates the clustering of activated receptors that is required for efficient particle binding.

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

Matrix vesicles (MVs), released by budding from apical microvilli of osteoblasts during bone formation and development, are involved in the initiation of mineralization by promoting the formation of hydroxyapatite in their lumen. To gain additional insights into MV 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 and compared. In total, 282 MV and 451 microvillar proteins were identified. Of those, 262 were common in both preparations, confirming that MVs originate from apical microvilli. The occurrence of vesicular trafficking molecules (e.g. Rab proteins) and of the on-site protein synthetic machinery suggests that cell polarization and apical targeting are required for the incorporation of specific lipids and proteins at the site of MV formation. MV release from 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.

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.

Interaction of annexin A6 with cholesterol rich membranes is pH-dependent and mediated by the sterol OH.

Annexin A6 (AnxA6), a calcium- and membrane-binding protein, is well-known to play a role in calcium homeostasis, membrane traffic and membrane organization. It had been suggested that, despite calcium-dependent interaction with anionic phospholipids, AnxA6 displays calcium-independent cholesterol binding properties. In this study, the following questions were addressed: does AnxA6 bind preferentially to cholesterol-containing biomimetic membranes? If so, what is the molecular mechanism of the binding? To answer these questions, human recombinant AnxA6-1 isoform was prepared and used with Langmuir monolayers containing various lipids. The interactions between AnxA6 and the lipid monolayers were examined by kinetic measurements of the interfacial adsorption and Brewster angle microscopy. We focused on the pH effect on the AnxA6 binding to monolayers containing cholesterol. At acidic pH, AnxA6-1 exhibits the highest affinity to monolayers containing the highest amount of cholesterol. Replacing cholesterol by cholesteryl acetate provided evidence that the hydroxyl group of cholesterol plays a role in AnxA6-lipid interactions. In addition, the affinity of recombinant AnxA6-1 tryptophan mutant (W343F) to the air/water interface and to lipid monolayers was tested. Substitution of Trp343 modified the interfacial properties of the protein and its interactions with sterol monolayers. Our results suggest that the linker region containing Trp343 is important for the interactions between AnxA6-1 and cholesterol.

Activated FcgammaRII and signalling molecules revealed in rafts by ultra-structural observations of plasma-membrane sheets.

To reveal topography of FcgammaRII components of the receptor-signalling complex, large plasma-membrane sheets were obtained by cell cleavage and analysed by immuno-electron microscopy. Non-activated FcgammaRII was dispersed in the plane of the plasma membrane and only rarely was localized in the proximity of Lyn, an Src family tyrosine kinase, and CD55, a glycosylphosphatidylinositol-anchored protein. After FcgammaRII activation by cross-linking with antibodies, clusters of an electron-dense material acquiring about 86% of FcgammaRII and reaching up to 300 nm in diameter were formed within 5 min. These structures also accommodated about 85% of Lyn and 63% of CD55 labels that were located in close vicinity of gold particles attributed to the cross-linked FcgammaRII . The electron-dense structures were also abundant in tyrosine phosphorylated proteins. At their margins PIP2 was preferentially located. Based on a concentration of Lyn, CD55 and activated FcgammaRII , the electron-dense structures seem to reflect coalescent membrane rafts.

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.

Calcium‐ and pH‐dependent localization of annexin A6 isoforms in Balb/3T3 fibroblasts reflecting their potential participation in vesicular transport

Annexin A6 (AnxA6), calcium‐ and membrane‐binding protein, is involved in membrane dynamics. It exists in the cell in two isoforms, AnxA6‐1 and AnxA6‐2, varying only by the VAAEIL sequence. In most cells, AnxA6‐1 predominates. A limited number of observations suggests that both isoforms differ from each other functionally. The EGF‐dependent Ca2+ influx in A431 cells is inhibited only by AnxA6‐1. Moreover, AnxA6‐2 was found to exhibit higher affinity for Ca2+. In this report we addressed the potential significance of the VAAEIL deletion in AnxA6‐2. For this purpose, we expressed AnxA6 isoform cDNAs in bacteria or mouse Balb/3T3 fibroblasts. The recombinant AnxA6‐2 was characterized by a less extended molecular shape than that of AnxA6‐1 and required a narrower [Ca2+] range to bind liposomes. Upon lowering pH in the presence of EGTA recombinant AnxA6‐2 became less hydrophobic than AnxA6‐1 as revealed by the Triton X‐114 partition. Furthermore, AnxA6‐2 revealed stronger F‐actin binding than that of AnxA6‐1. Immunofluorescence microscopy showed that the EGFP‐tagged AnxA6 isoforms expressed in Balb/3T3 fibroblasts relocate in a Ca2+‐ and H+‐sensitive manner to the vesicular structures in a perinuclear region or in cytosol. Cell fractionation showed that in resting conditions AnxA6‐1 is associated with early endosomes and AnxA6‐2 with late endosomes, and an increase in [Ca2+] and/or [H+] induced their opposite distribution. These findings suggest a potentially independent regulation, localization, and function of AnxA6 isoforms in Balb/3T3 fibroblasts. More generally, our findings suggest distinct functions of AnxA6 isoforms in membrane dynamics. J. Cell. Biochem. 104: 418–434, 2008.

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.