Bone biomechanical properties in prostaglandin EP1 and EP2 knockout mice
Introduction
Prostaglandins are strong modulators of bone metabolism and their administration to rodents results in increased cancellous and cortical bone mass translating into enhanced mechanical strength of the skeleton.35 Among the many prostaglandins, the E series, prostaglandin E2 (PGE2), has proven to be a potent systemic1, 14, 15, 16, 17, 18, 19, 23, 24, 27, 37, 41 and local bone anabolic agent.30, 43 Treatment with PGE2 results in substantial production of new bone manifested by increased osteoblast numbers and labeled surfaces in animal,14, 18, 19, 23 and human20 studies. In addition, PGE2 is known to have a biphasic effect on osteoblast cell lines9, 28, 29 with cell differentiation seen at low concentrations and cell proliferation seen at high concentrations. However, the mechanism of action of PGE2 on bone tissue is not fully understood and is still under investigation.
PGE2 has multiple effects on biological activities in bone and is mediated through various receptors. For example, PGE2 stimulates bone formation and resorption through osteoblastic and osteoclastic activities, respectively. The effects of PGE2 are mediated by four well-characterized receptors (EP1– EP4).4, 7, 10, 13, 22, 25, 26, 32, 34, 39, 42 These PGE2 receptors have been cloned and phenotyped in humans,4, 22 rats,32 dogs,11 and mice.13, 34, 42 Each EP receptor has a unique intracellular signaling pathway in bone. For instance, EP1 is coupled with intracellular Ca2+ transport, whereas EP3 blocks cellular adenylate cyclase.21, 34, 42 On the other hand, EP2 and EP4 receptors stimulate cellular adenylate cyclase to accumulate cellular cAMP in osteoblast and osteoclast cells,25, 26, 31, 36, 39, 44 thus affecting bone metabolism. Similar to EP2 the EP4 receptor also plays a major role in stimulating bone resorption through increased osteoclast numbers as observed in mouse calvaria cultures.31, 36
Recent in vitro data8 from young rat calvaria cells suggest that the PGE2-related effects on osteoblast proliferation and differentiation (resulting bone nodules) were mediated through the EP1 receptor. PGE2 enhanced bone nodule formation and alkaline phosphatase (ALP) activity in osteoblast cells mediated through EP1 receptors.8 On the other hand, PGE2 analogs, acting through the EP2/EP4 receptor, had an opposite effect on osteoblast cells, resulting in reduced bone nodule formation and ALP activity but enhanced cell proliferation.8 In addition, PGE2 also affects other biological systems through the EP2 receptor, causing decreased blood pressure and low fertility in female EP2 knockout mice.38 Absence of either of these receptors (EP1 and EP2) may interrupt specific PGE2 pathways and thus directly or indirectly affect bone physical and mechanical properties.
This study compares bone physical and biomechanical properties in EP1 and EP2 knockout mice to their corresponding wild-type controls. We examined both axial (vertebrae) and appendicular (femurs, ulnae) bones from adult mice.
Section snippets
Materials and methods
We examined bone biomechanical properties in two sets of adult (4 months of age) female mice. These included: (a) EP1 knockout (n = 12), and wild-type control (DBAl/lacJ) (n = 13) animals;38 and (b) mice heterozygous for a targeted disruption of the EP2 gene38 crossed with C57BL/6 × DBA/2 F1 then intercrossed to produce homozygous EP2 knockout (n = 20) and wild-type control (n = 21) animals. At autopsy, the femurs, vertebral bodies, and ulnae were collected and frozen in saline at −20°C for
Results
The physical properties, including body weight, femoral length, femoral diaphysis total cross-sectional area, marrow area, cortical area, second moment of area, ulnar dry and ash weights, and vertebral body height were not different between the EP1 knockout and corresponding wild-type control mice (Table 1). However, the vertebral body cross-sectional area in the EP1 knockout mice was greater than in wild-type controls. Body weight, diaphyseal total area, and marrow area in the femurs and
Discussion
We investigated the action of PGE2 receptors, EP1 and EP2, on the physical and biomechanical properties of ulnae, femurs, and vertebral bodies from EP1 and EP2 knockout mice by comparing them with their corresponding wild-type controls. The majority of the physical (except for the vertebral body cross-sectional area) and biomechanical properties of the EP1 knockout and corresponding wild-type controls were similar. Although most of the values for physical properties were higher, the
Conclusions
This study has found that EP1 receptors are not essential for skeletal strength but do have some site-specific effect on bone size. The absence of EP1 receptors resulted in greater vertebral cross-sectional area in EP1 knockout mice as compared with wild-type control mice. We also found that EP2 receptors have a major influence on the biomechanical properties of bone in mice. The absence of EP2 receptors resulted in weak bone biomechanical strength properties and greater bone size in the EP2
Acknowledgements
This work was supported by NIH Grant 12556. The authors thank Dr. Lydia Pan (Pfizer, Inc., Central Research, Groton, CT) for providing all of the mice used in these studies. We also thank Amy Peyton and Mark Covey for assistance in specimen collection and preparation for testing.
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