Human apolipoprotein E isoforms differentially affect bone mass and turnover in vivo

Abstract

The primary role of apolipoprotein E (apoE) is to mediate the cellular uptake of lipoproteins. However, a new role for apoE as a regulator of bone metabolism in mice has recently been established. In contrast to mice, the human APOE gene is characterized by three common isoforms APOE ε2, ε3 and ε4 that result in different metabolic properties of the apoE isoforms, but it remains controversial whether the APOE polymorphism influences bone traits in humans. To clarify this, we investigated bone phenotypes of apoE knock-in mice, which express one human isoform each (apoE2 k.i., apoE3 k.i., apoE4 k.i.) in place of the mouse apoE. Analysis of 12 week-old female knock-in mice revealed increased levels of biochemical bone formation and resorption markers in apoE2 k.i. animals as compared to apoE3 k.i. and apoE4 k.i., with a reduced OPG/RANKL ratio in apoE2 k.i., indicating increased turnover with prevailing resorption in apoE2 k.i.. Accordingly, histomorphometric and μCT analyses demonstrated significantly lower trabecular bone mass in apoE2 than in apoE3 and apoE4 k.i. animals, which was reflected by a significant reduction of lumbar vertebrae maximum force resistance. Unlike trabecular bone, femoral cortical thickness, and stability was not differentially affected by the apoE isoforms. To extend these observations to the human situation, plasma from middle-aged healthy men homozygous for ε2/ε2, ε3/ε3, and ε4/ε4 (n=21, n=80, n=55 respectively) was analyzed with regard to bone turnover markers. In analogy to apoE2 k.i. mice, a lower OPG/RANKL ratio was observed in the serum of ε2/ε2 carriers as compared to ε3/ε3 and ε4/ε4 individuals (p=0.02 for ε2/ε2 vs ε4/ε4). In conclusion, the current data strongly underline the general importance of apoE as a regulator of bone metabolism and identifies the APOE ε2 allele as a potential genetic risk factor for low trabecular bone mass and vertebral fractures in humans.

Apolipoprotein E isoforms and bone—of mice and men

“…(4,5) Although many association studies have been performed in various populations, and evidence has been sought for an association between ApoE isoforms and bone phenotypes, the results have been conflicting, probably because the studies have been underpowered to detect the very modest effects on bone mineral density (BMD), bone turnover markers, and fracture that would be expected for variants of this type. (6) For example, in some studies, trends were observed for reduced BMD and increased fracture risk in carriers of the ApoE4 allele, (4,7,8) which is the opposite of the results reported by Deickmann and colleagues (1) in mice; however, in other studies, no associations between ApoE alleles and BMD or fractures were observed. (9,10) Moreover, in a recent meta-analysis of genome-wide association studies that involved more than 100,000 subjects, the ApoE locus did not emerge as a significant determinant of BMD or fracture.…”

“…(1) The preclinical experiments were performed using genetically engineered mice in which the endogenous coding sequences of ApoE had been replaced with one of the human ApoE2, ApoE3, or ApoE4 isoforms (so called knock-in mice). These three isoforms differ from each other in the amino acid sequence at codons 118 and 158.…”

“…Deickmann and colleagues (1) report that different isoforms of the human Apolipoprotein E gene (APOE) significantly influence bone turnover and bone mass in mice, and they present some data to suggest the same might be true in man. (1) The preclinical experiments were performed using genetically engineered mice in which the endogenous coding sequences of ApoE had been replaced with one of the human ApoE2, ApoE3, or ApoE4 isoforms (so called knock-in mice).…”

“…APOE has also been implicated in the pathogenesis of Alzheimer's disease, which is strongly associated with carriage of ApoE4; however, the mechanisms by which the ApoE4 variant predisposes to Alzheimer's are incompletely understood. (2) The experiments of Deickmann and colleagues (1) were prompted by the observation that deletion of ApoE in mice is associated with high bone mass due to increased bone formation (3) and by genetic association studies indicating that common polymorphic variants of ApoE may be genetic risk factors for osteoporosis and fragility fractures. (4,5) Although many association studies have been performed in various populations, and evidence has been sought for an association between ApoE isoforms and bone phenotypes, the results have been conflicting, probably because the studies have been underpowered to detect the very modest effects on bone mineral density (BMD), bone turnover markers, and fracture that would be expected for variants of this type.…”

“…Only one previous study used a similar approach to that of Deickmann and colleagues, (1) and that study found no difference in BMD values or biochemical markers of bone turnover in ApoE2 homozygotes (n ¼ 18) as opposed to ApoE4 homozygotes (n ¼ 18). (11) No doubt, however, promoted by Deickmann and colleagues' (1) results, other investigators who have studied these polymorphisms in reasonably large numbers of individuals (9,10) may go back to their original data to determine if they can detect a significant association between ApoE2 homozygosity and BMD or biochemical markers of bone turnover.Although the data of Deickmann and colleagues (1) clearly show that human ApoE2 does influence bone mass and bone turnover when expressed in the homogeneous genetic background of a knock-in mouse, the situation is rather more complex in humans, where hundreds of genetic variants contribute to the regulation of bone mass and bone turnover. (7) Although one cannot exclude the possibility that common ApoE isoforms play a role in regulating bone metabolism in man, it seems probable that they do not have a large enough effect size for associations to be consistently observed in human studies.…”