Date Published: March 17, 2015
Publisher: Public Library of Science
Author(s): Shilpa Choudhary, Ernesto Canalis, Thomas Estus, Douglas Adams, Carol Pilbeam, Pierre J Marie.
We previously reported that the ability of continuously elevated PTH to stimulate osteoblastic differentiation in bone marrow stromal cell cultures was abrogated by an osteoclastic factor secreted in response to cyclooxygenase-2 (Cox2)-produced prostaglandin E2. We now examine the impact of Cox2 (Ptgs2) knockout (KO) on the anabolic response to continuously elevated PTH in vivo. PTH (40 μg/kg/d) or vehicle was infused for 12 or 21 days in 3-mo-old male wild type (WT) and KO mice in the outbred CD-1 background. Changes in bone phenotype were assessed by bone mineral density (BMD), μCT and histomorphometry. PTH infusion for both 12 and 21 days increased femoral BMD in Cox2 KO mice and decreased BMD in WT mice. Femoral and vertebral trabecular bone volume fractions were increased in KO mice, but not in WT mice, by PTH infusion. In the femoral diaphysis, PTH infusion increased cortical area in Cox2 KO, but not WT, femurs. PTH infusion markedly increased trabecular bone formation rate in the femur, serum markers of bone formation, and expression of bone formation-related genes, growth factors, and Wnt target genes in KO mice relative to WT mice, and decreased gene expression of Wnt antagonists only in KO mice. In contrast to the differential effects of PTH on anabolic factors in WT and KO mice, PTH infusion increased serum markers of resorption, expression of resorption-related genes, and the percent bone surface covered by osteoclasts similarly in both WT and KO mice. We conclude that Cox2 inhibits the anabolic, but not the catabolic, effects of continuous PTH. These data suggest that the bone loss with continuously infused PTH in mice is due largely to suppression of bone formation and that this suppression is mediated by Cox2.
Parathyroid hormone (PTH) is a major systemic regulator of calcium homeostasis and bone turnover. PTH acts on a G-protein coupled receptor, PTH1R, expressed on osteoblast lineage cells to stimulate bone formation via Gαs/cAMP-activated pathways [1–4]. Intermittent PTH was the first anabolic agent approved for osteoporosis therapy in the USA [5,6]. When PTH is injected intermittently, both bone formation and resorption are increased but formation is increased more than resorption. On the other hand, when PTH levels are continuously elevated, resorption is greater than formation and bone is lost [7,8]. It is unclear if this change in the bone turnover from net formation to net resorption is due to increased resorption with continuous PTH, compared to intermittent PTH, or if it is the formation response that is reduced. A number of in vitro studies report that continuous PTH treatment inhibits osteoblast differentiation [3,9–11], and some in vivo studies conclude that continuous PTH infusion suppresses bone formation . These observations suggest that the bone loss associated with continuous PTH infusion is not simply the result of increased resorption but may involve suppressed bone formation.
We used adult male mice at 3-months of age for studies to avoid the rapid skeletal growth phase that might make effects of PTH difficult to interpret. The dose and duration of PTH infusion were chosen based on earlier studies showing the catabolic effects of continuous PTH infusion [7,8].
In Cox2 KO mice, PTH infusion was anabolic increasing femoral BMD, serum markers of bone formation, femoral and vertebral trabecular bone volume, cortical area, % osteoblast surface, bone formation rate, and expression of genes thought to reflect or mediate increased bone formation. In WT mice, on the other hand, the overall effect of PTH infusion was catabolic. PTH decreased femoral BMD in WT mice in both 12 and 21 day infusions and decreased BV/TV and Tb.N in all μCT analyses of femoral and vertebral bone from the 12 and 21 day infusions, although the decrease was statistically significant only in the femur in the 21 day infusion. Tb.Sp was significantly increased by PTH in three of the four analyses. However, PTH did stimulate small increases in serum P1NP, % osteoblast surface, bone formation rate, and Osteocalcin gene expression in the 12 day infusion. Otherwise, there was little or no effect on expression of genes thought to mediate PTH anabolic effects in WT mice. These results are similar to those seen in other studies of continuous PTH in WT mice [32,39], despite marked differences in PTH dose, mouse age, gender or background, and are consistent with PTH stimulating both resorption and formation but stimulating resorption more than formation because formation is suppressed.