Abstract
While the basic cellular contributions to bone differentiation and mineralization are widely accepted, the regulation of these processes at the intracellular level remains inadequately understood. Our laboratory recently identified annexin 2 as a protein involved in osteoblastic mineralization. Annexin 2 was overexpressed twofold in SaOSLM2 osteoblastic cells as a fusion protein with green fluorescent protein. The overexpression of annexin 2 led to an increase in alkaline phosphatase activity as well as an increase in mineralization. Our data suggest that the increase in alkaline phosphatase activity does not result from increased alkaline phosphatase transcript or protein levels; therefore we evaluated mechanism of action. We determined that both annexin 2 and alkaline phosphatase activity were localized to membrane microdomains called lipid rafts in osteoblastic cells. Annexin 2 overexpression resulted in an increase in alkaline phosphatase activity that was associated with lipid microdomains in a cholesterol-dependent manner. Furthermore, disruption of lipid rafts with a cholesterol sequestering agent or reduction of annexin 2 expression by specific antisense oligonucleotides each resulted in diminished mineralization. Therefore, intact lipid rafts containing annexin 2 appear to be important for alkaline phosphatase activity and may facilitate the osteoblastic mineralization process.
Highlights
Bone is a dynamic tissue that is replaced throughout life
We have shown that anx2 is not localized to the nucleus or the cell surface; rather it is restricted to the cytoplasm and the intracellular aspect of the plasma membrane where it may interact with lipid microdomains
Overexpression of anx2 in osteoblastic cells In order to determine the potential role of anx2 in osteoblastic cells, we developed an anx2GFP fusion construct that was stably transfected into SaOSLM2 osteoblastic cells
Summary
The normal process of bone turnover requires several levels of communication between the osteoblasts, which produce bone, and the osteoclasts, which resorb it. Osteoblasts have the primary function of making and secreting osteoid, the extracellular matrix (ECM) and associated proteins required for mineralization. A variety of proteins have been implicated in the bone mineralization process including collagen I and alkaline phosphatase (ALP). ALP catalyzes the hydrolysis of phosphomonoesters at an alkaline pH and breaks down pyrophosphate, which is an important inhibitor of calcium phosphate deposition at the extracellular level (Johnson et al, 2000; Miao and Scutt, 2002). Four isozymes of ALP have been identified in humans: tissue non-specific liver/bone/kidney, intestinal, placental, and germ cell ALP (Miao and Scutt, 2002). Regulation of the localization and activity of ALP and other proteins is important in the ultimate control of bone differentiation and mineralization
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