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. 2021 Apr 13;22(8):3993.
doi: 10.3390/ijms22083993.

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

Lukasz Bozycki  1 , Joanna Mroczek  1   2 , Laurence Bessueille  3   4   5   6   7 , Saida Mebarek  3   4   5   6   7 , René Buchet  3   4   5   6   7 , Slawomir Pikula  1 , Agnieszka Strzelecka-Kiliszek  1
Affiliations

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

Lukasz Bozycki et al. Int J Mol Sci. .

Abstract

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.

Keywords: Saos-2 osteosarcoma cells; annexins; fetuin-A; hFOB 1.19 osteoblastic cells; matrix vesicles; mineralization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Morphology of hFOB 1.19 (A,C) and Saos-2 (B,D) cells in resting (A,B) and stimulated (C,D) conditions. Longest cell axis of cells in resting conditions (white) or after seven-day stimulation with ascorbic acid (AA) and β-glycerophosphate (β-GP) (red) observed under an Axiovert 40C light microscope (Carl Zeiss, Poznan, Poland) with Phase contrast, magnification at 120×. Arrows indicate an elongated cell morphology, whereas the arrowhead—a shortened cell axis. (E) In total, 500 cells were analyzed: 10 photographs were taken at each of five random locations of a 100 mm diameter dish, and the longest axis of 10 cells at each location was measured for each cell variant using Image J bundled with 64-bit Java 2.8.0_112 software (Bethesda, MD, USA) and presented as the fold of change of the longest axis of hFOB 1.19 resting cells; * p < 0.05.
Figure 2
Figure 2
Mineralization level of hFOB 1.19 (A,C) and Saos-2 (B,D) cells in resting conditions (white) or after seven-day stimulation with AA and β-GP (red). Cells were either non-treated (−) or treated (+) with inhibitors: 100 μM levamisole (Lev) for Tissue non-specific alkaline phosphatase (TNAP) activity or 25 μM K201 for Ca channel activity. Ca salts (A,B) were stained with alizarin red-S (AR-S) dissolved in cetyl pyridinium chloride (CPC), and their content was measured spectrophotometrically at λ 562 nm. TNAP activity (C,D) was measured using alkaline phosphatase (ALP) Yellow para-nitro phenyl phosphate (pNPP) Liquid Substrate System for ELISA (Sigma-Aldrich, Warsaw, Poland), and absorbance was recorded spectrophotometrically at λ 405 nm. Data are means ± s.e. of at least three experiments; * p < 0.05 and ** p < 0.01.
Figure 3
Figure 3
IR spectra of minerals formed by vesicular fractions of hFOB 1.19 (A) and Saos-2 (B) cells. Vesicular fractions were purified from the extracellular matrix (MVs) and from the cytoplasm (CVs) of collagenase-treated cells in resting conditions (R) or after stimulation with AA and β-GP (S). The vesicular fractions were dried. IR spectrum of apatite (HA) is characterized by peaks 1022, 633, 600, and 559 cm−1. The red rectangle indicates the presence of HA in the fraction of MVs derived from the Saos-2 cell line stimulated for mineralization. IR spectra were averaged from three independent samples, each measured at two distinct locations of the batch (n = 6). IR spectra were up scaled by 0.2 A to be better visible.
Figure 4
Figure 4
Co-localization of AnxA6, AnxA2, or fetuin-A (FetuA) with TNAP in hFOB 1.19 (A,C,E) and Saos-2 (B,D,F) cells in resting conditions (R) or after seven-day stimulation with AA and β-GP (S). The cells were incubated with appropriate antibodies: mouse monoclonal anti-AnxA6 (A,B), mouse monoclonal anti-AnxA2 (C,D), or mouse monoclonal anti-FetuA (E,F), all followed by goat anti-mouse IgG-FITC (green); rabbit polyclonal anti-TNAP followed by goat anti-rabbit IgG-TRITC (red) and DAPI for nuclei (blue) and observed under an Axio Observer.Z1 FM (Carl Zeiss, Poznan, Poland) with Phase contrast and appropriate fluorescent filters, magnification 630 x. Arrowheads indicate protein accumulation in vesicular and/or cluster structures. The yellow color and arrows on the merge images indicate AnxA6 (A,B), AnxA2 (C,D), or FetuA (E,F) co-localization with TNAP. Results of a typical experiment are presented.
Figure 5
Figure 5
Co-localization of AnxA6 with TNAP in hFOB 1.19 (A,C) and Saos-2 (B,D) cells after treatment with 100 μM levamisole (A,B) or 25 μM K201 (C,D) in resting conditions (R) or after seven-day stimulation with AA and β-GP (S). The cells were incubated with appropriate antibodies: mouse monoclonal anti-AnxA6 followed by goat anti-mouse IgG-FITC (green), rabbit polyclonal anti-TNAP followed by goat anti-rabbit IgG-TRITC (red) and DAPI for nucleus (blue) and observed under an Axio Observer.Z1 FM (Carl Zeiss, Poznan, Poland) with Phase contrast and appropriate fluorescent filters, magnification at 630×. Arrowheads indicate protein accumulation in vesicular and/or cluster structures. The yellow color and arrows on the merge images indicate AnxA6 co-localization with TNAP. Results of a typical experiment are presented.
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References

    1. Aghajanian P., Mohan S. The art of building bone: Emerging role of chondrocyte-to-osteoblast transdiferentiation in endochondral ossification. Bone Res. 2018;6:19. doi: 10.1038/s41413-018-0021-z. - DOI - PMC - PubMed
    1. Anderson H.C. Matrix vesicles and calcification. Curr. Rheumatol. Rep. 2003;5:222–226. doi: 10.1007/s11926-003-0071-z. - DOI - PubMed
    1. Bottini M., Mebarek S., Anderson K.L., Strzelecka-Kiliszek A., Bozycki L., Simão A.M.S., Bolean M., Ciancaglini P., Pikula J.B., Pikula S., et al. Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models. Biochim. Biophys. Acta (BBA) Gen. Subj. 2018;1862:532–546. doi: 10.1016/j.bbagen.2017.11.005. - DOI - PMC - PubMed
    1. Golub E.E. Role of matrix vesicles in biomineralization. Biochim. Biophys. Acta (BBA) Gen. Subj. 2009;1790:1592–1598. doi: 10.1016/j.bbagen.2009.09.006. - DOI - PMC - PubMed
    1. Wuthier R.E., Lipscomb G.F. Matrix vesicles: Structure, composition, formation and function in calcification. Front. Biosci. 2011;16:2812–2902. doi: 10.2741/3887. - DOI - PubMed

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