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The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptormediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-frans-retinoic acid (RA) in T-47D human breast cancer cells and that RAtreated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells.

Discussion

Megalin is a large transmembrane protein (600 kDa), which, together with its endocytic partners cubilin and Dab2, is essential for renal uptake of the major circulating form of calciferols (25D3) (4,5,8). Practically all 25D3 (>99%) circulates and is internalized as an intact 25D3-DBP complex. Once internalized by renal proximal tubule cells, the vitamin-carrier complex dissociates, allowing for metabolic activation of 25D3 into 1,25D3, the active vitamin D metabolite that binds the vitamin D receptor, which mediates ant i proliferative and prodifferentiating signaling in a number of extra-renal tissues (27). Hence, considerable attention has been focused on whether tissues other than the kidney can generate 1,25D3. In support of this concept, the enzyme capable of converting 25D3 to 1,25D3 (CYP27B1) has now been conclusively identified in many additional cell types, including keratinocytes, colonocytes, and the epithelial cells of the prostate and mammary gland (16, 28,29). Although the presence of CYP27B1 in extra-renal tissues suggests that local activation of 25D3 can occur, this suggestion presumes that circulating 25D3 is accessible to the enzyme. Thus, determining the mechanisms by which the 25D3-DBP complex can be internalized by extra-renal tissues could be a critical step in determining why serum 25D3 levels are inversely correlated with incidence of numerous types of cancer.

Although administration of 1,25D3 has been proven to be effective at inhibiting tumorigenesis, 1,25D3 produces a toxic calcémie response when administered at pharmacological doses (34). In addition, strict regulation of renal 1,25D3 synthesis ensures that under physiological conditions, serum 1,25D3 concentrations will not reach levels that can inhibit tumor growth regardless of the amount of vitamin D acquired from the diet (35). Therefore, evaluating the ability of mammary cells to locally produce 1,25D3 may be more predictive of breast cancer risk and may lead to a more feasible strategy for the use of dietary vitamin D in the context of cancer prevention. In support of this concept, epidemiological studies report that both sunlight exposure and dietary vitamin D are inversely correlated with breast cancer risk or disease progression (36-40). Rats fed diets low in vitamin D develop significantly more mammary tumors when treated with chemical carcinogens than rats with adequate vitamin D status (41). Furthermore, women with suboptimal vitamin D status exhibited increased mammographie breast density (42). Taken together, this evidence suggests that determination of uptake mechanisms and tissue stores of vitamin D in the mammary gland is a critical step toward understanding the role of dietary or sunlight-derived vitamin D in breast cancer prevention.