Expression of LRP1 by human osteoblasts: a mechanism for the delivery of lipoproteins and vitamin K1 to bone.
Accumulating clinical and experimental data show the importance of dietary lipids and lipophilic vitamins, such as vitamin K1, for bone formation. The molecular mechanism of how they enter the osteoblast is unknown. Here we describe the expression of the multifunctional LRP1 by human osteoblasts in vitro and in vivo. We provide evidence that LRP1 plays an important role in the uptake of postprandial lipoproteins and vitamin K1 by human osteoblasts.


BACKGROUND
Chylomicrons (CM) and their remnants (CR) represent the postprandial plasma carriers of dietary lipids. Dietary vitamin K1 is known to be transported in the circulation as part of CM/CR and is required by osteoblasts as an essential co-factor for the gamma-carboxylation of bone matrix proteins. The molecular mechanisms underlying the delivery of lipophilic substances to bone are not understood. In this study, the expression and function of CM/CR receptors was examined in human osteoblasts.


METHODS
Four human osteoblast-like cell lines were analyzed: two osteosarcoma lines (MG63, SaOS-2) and two telomerase-immortalized human bone marrow stromal cell lines (hMSC-TERT [4] and [20]) after 1,25(OH)2 vitamin D3 induction of osteoblastic differentiation (hMSC-TERT-OB). Receptor expression was examined by Western blotting and immunohistochemistry of normal human bone sections. Endocytotic receptor function was analyzed by cellular uptake assays using fluorescent and radiolabeled human CR. Vitamin K1-enriched CR (CR-K1) were generated in vivo after oral vitamin administration and vitamin K1 uptake by osteoblasts was measured by HPLC. The effect of CR-K1 uptake on osteocalcin carboxylation was measured by ELISA.


RESULTS
Osteoblasts exhibit high levels of protein expression of the CR receptors LRP1 and LDLR. VLDLR is expressed to a lower degree. Immunohistochemistry of normal human bone sections showed strong LRP1 expression by osteoblasts and marrow stromal cells. Uptake of fluorescent CR by osteoblasts resulted in the typical pattern of receptor-mediated endocytosis. CR uptake was stimulated by the exogenous addition of the lipoprotein receptor ligands apolipoprotein E and lipoprotein lipase. Uptake was reduced by the known LRP1 inhibitors RAP, lactoferrin, and suramin, but not by LDL, which exclusively binds to the LDLR. Vitamin K1 uptake by hMSC-TERT-OB after incubation with CR-K1 was also shown to be sensitive to LPL stimulation and the LRP1 specific inhibitor lactoferrin. CR-K1 uptake into osteoblasts stimulated the gamma-carboxylation of osteocalcin.


CONCLUSIONS
Human osteoblasts express receptors of the LDLR family with a capacity for vitamin K1 uptake through CR endocytosis, a novel mechanism for the delivery of dietary lipids and lipophilic vitamins to human bone. The current data suggest that, among the expressed receptors, LRP1 plays a predominant role.
Niemeier
Andreas
A
Department of Orthopaedics, University Hospital Hamburg-Eppendorf, Hamburg, Germany. niemeier@uke.uni-hamburg.de
Kassem
Moustapha
M
Toedter
Klaus
K
Wendt
Dorte
D
Ruether
Wolfgang
W
Beisiegel
Ulrike
U
Heeren
Joerg
J
D000284:Administration, Oral; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D001842:Bone and Bones / Q000378:metabolism*; D045744:Cell Line, Tumor; D002762:Cholecalciferol / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002914:Chylomicrons / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004797:Enzyme-Linked Immunosorbent Assay; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D007150:Immunohistochemistry; D007781:Lactoferrin / Q000378:metabolism; D008024:Ligands; D050356:Lipid Metabolism; D008071:Lipoprotein Lipase / Q000378:metabolism; D008074:Lipoproteins / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D018613:Microscopy, Confocal; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000378:metabolism; D011485:Protein Binding; D011973:Receptors, LDL / Q000096:biosynthesis*; D018110:Receptors, Lipoprotein / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology; D013997:Time Factors; D014812:Vitamin K / Q000378:metabolism; D010837:Vitamin K 1 / Q000378:metabolism*
D002914:Chylomicrons; C434476:LRP1B protein, human; D008024:Ligands; D008074:Lipoproteins; D011973:Receptors, LDL; D018110:Receptors, Lipoprotein; C028203:chylomicron receptor; D015675:Osteocalcin; D014812:Vitamin K; D002762:Cholecalciferol; D010837:Vitamin K 1; D008071:Lipoprotein Lipase; D007781:Lactoferrin