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First published online 16 December 2003
doi: 10.1242/jcs.00909

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The role of annexin 2 in osteoblastic mineralization

Jennifer M. Gillette¹ and Sheila M. Nielsen-Preiss^2,*,

¹ Department of Cellular and Developmental Biology, University of Colorado Health Sciences Center, Denver, CO 80262, USA
² Department of Orthopaedics, University of Colorado Health Sciences Center, Denver, CO 80262, USA

View larger version (35K): [in a new window]   Fig. 1. Anx2 overexpression in osteoblastic cells. SaOSLM2 cells were stably transfected with GFP or an anx2GFP fusion construct. (A) Immunoblot analysis confirmed that endogenous anx2 was expressed similarly in the GFP (1) and the anx2GFP (2) transfectants and the exogenous fusion protein was expressed in the transfectants. Endogenous anx2 was detected at approximately 36 kDa whereas the fusion protein was shifted to approximately 63 kDa because of the 27 kDa GFP tag. (B) Epifluorescence microscopy of GFP reveals that the fusion protein is excluded from the nucleus, similarly to endogenous anx2 protein expression (unpublished results) (200x).

View larger version (101K): [in a new window]   Fig. 2. Anx2 overexpression enhances osteoblastic mineralization. A panel of cell cultures were maintained under normal conditions (A,C,E) or under conditions to induce differentiation (B,D,F). All cells were then stained with Alizarin Red S to detect insoluble calcium nodules and analyzed at 40x magnification.

View larger version (18K): [in a new window]   Fig. 3. Anx2 overexpression results in an increase in ALP activity. (A) ALP protein was detected by immunofluorescence in SaOSLM2 cells overexpressing anx2. (B) Cell-associated ALP activity was measured in osteoblast-like cells cultured in both non-differentiation-(white bars) and differentiation-inducing (black bars) media. * Statistically significant increases in ALP activity by culturing the cells under differentiation conditions (P<0.05). #Statistically significant increases in ALP activity induced by anx2 overexpression (P<0.05).

View larger version (54K): [in a new window]   Fig. 4. Anx2 overexpression does not alter ALP gene expression levels. (A) 1 µg of total RNA from SaOSLM2 (A) or SaOSLM2-Anx2GFP (B) cells was subjected to semi-quantitative RT-PCR analysis of ALP or G3PDH message levels. RNA was isolated from cells plated for 0.5, 1.0, 1.5 and 2.0 weeks. Band intensities for ALP signal from GFP-(C) and anx2GFP-(D) expressing cells were normalized against those obtained for G3PDH in the same samples (E,F).

View larger version (17K): [in a new window]   Fig. 5. Osteoblastic cells contain detergent insoluble anx2. (A) Solublization of U2OS cells in 1% Triton X-100 results in a soluble fraction of anx2 (S), and an insoluble fraction of anx2 (P). (B) Cellular lysates were made from both SaOSLM2-anx2GFP-(1) and GFP-(2) expressing cells. Conditioned medium (CM) was also collected from these cells. (C) The membranous fraction following subcellular fractionation of SaOSLM2 cells was treated with 0.01% digitonin or the diluent methanol for 10 minutes. Membranes were resuspended and these insoluble membrane pellets (P) as well as the soluble wash (W) components were analyzed by immunoblot for the presence of anx2.

View larger version (17K): [in a new window]   Fig. 6. Anx2 and ALP are associated with lipid rafts. (A) Lipid raft microdomains were isolated from SaOSLM2 and SaOSLM2-anx2GFP cells by centrifugation through a 5-40% sucrose gradient. Fractions were collected and insoluble material was pelleted from each fraction in which the sucrose had been reduced. Anx2 was detected by immunoblot analysis. (B) ALP activity was evaluated in the lipid raft fraction pellets isolated from SaOSLM2 (solid line) and SaOSLM2-anx2GFP (dashed line) cells. The * in fraction 6 represents statistical significance between anx2-overexpressing ALP activities and parental ALP activities (P<0.05).

View larger version (39K): [in a new window]   Fig. 7. Cells cultured in the presence of a cholesterol-sequestering agent have a reduced ability to mineralize. SaOSLM2 cells (A) were cultured in conditions to induce mineralization in the presence or absence of 0.5 mM methyl-ß-cyclodextrin and stained with Alizarin Red S. The Alizarin Red S was solubilized in 10% cetylpyridinium chloride and quantitated by absorbance at 575 nm (B). OD values were normalized to that obtained from cells maintained in normal conditions.

View larger version (25K): [in a new window]   Fig. 8. Reduced anx2 expression in SaOSLM2 cells diminishes alkaline phosphatase activity and mineral crystal formation. (A) SaOSLM2 cells were transfected with an anx2-specific antisense oligonucleotide (1) or a control oligonucleotide (2). (B,C) Anx2 expression was analyzed by western blot at day 4. SaOSLM2 cells with either the anx2-specific oligonucleotide (Anx2AS) or the standard oligonucleotide (Std) were exposed to conditions to induce mineralization for 4 days. The cultures were stained with Alizarin Red S to detect deposition of mineral crystal (200x). (D) ALP activities were also measured in the Anx2AS (black) and the Std (white) oligonucleotide-treated cells.