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.

A novel retinoid binding property of human annexin A6.

Vitamin A (all-trans retinol) and all-trans retinoid acid (ATRA) interacted with human annexin A6 (AnxA6) as evidenced by AnxA6-induced blue shift of retinoid absorption maxima, by AnxA6-Trp fluorescence quenching and by a fluorescence resonance energy transfer from a Trp residue of AnxA6 to retinol. In addition, both retinoids stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. Up to our knowledge, it is a first report supporting the hypothesis of a direct implication of AnxA6 in vitamin A-dependent tissue mineralization.

Cholesterol modulates the membrane binding and intracellular distribution of annexin 6.

Annexins are Ca(2+)- and phospholipid-binding proteins that are widely expressed in mammalian tissues and that bind to different cellular membranes. In recent years its role in membrane traffic has emerged as one of its predominant functions, but the regulation of its intracellular distribution still remains unclear. We demonstrated that annexin 6 translocates to the late endocytic compartment in low density lipoprotein-loaded CHO cells. This prompted us to investigate whether cholesterol, one of the major constituents of low density lipoprotein, could influence the membrane binding affinity and intracellular distribution of annexin 6. Treatment of crude membranes or early and late endosomal fractions with digitonin, a cholesterol-sequestering agent, displayed a strong reduction in the binding affinity of a novel EDTA-resistant and cholesterol-sensitive pool of annexin 6 proteins. In addition, U18666A-induced accumulation of cholesterol in the late endosomal compartment resulted in a significant increase of annexin 6 in these vesicles in vivo. This translocation/recruitment correlates with an increased membrane binding affinity of GST-annexin 6 to late endosomes of U18666A-treated cells in vitro. In conclusion, the present study shows that changes in the intracellular distribution and concentration of cholesterol in different subcellular compartments participate in the reorganization of intracellular pools of Ca(2+)-dependent and -independent annexin 6.