Deletion of the Parathyroid Hormone Receptor in Marrow Adipose Lineage Precursors (MALPs) Prevents Their Negative Regulation of Skeletal Homeostasis

Abstract

Background and objectives: Parathyroid hormone (PTH) is essential for skeletal homeostasis and PTH[1-34] (teriparatide) is used to treat severe osteoporosis, medication-related osteonecrosis of the jaws, and enhance implant osseointegration. PTH exerts its anabolic actions by acting on osteoblasts, bone lining cells, and osteocytes. Recently, marrow adipose lineage precursors (MALPs) have been reported to suppress osteogenesis and enhance osteoclastogenesis. We hypothesized that, since MALPs express the PTH receptor 1 (Pth1r), they may contribute to the skeletal response to PTH. Methods: We deleted Pth1r specifically in MALPs and their lineage (adipocytes) using Adiponectin Cre (AdipoqCre) and tdTomato was used as a reporter. AdipoqCre;Pth1rfl/fl,tdTomatofl/fl mice (Pth1rMALPs) were used as experimental mice and age- and sex-matched AdipoqCre;Pth1rfl/fl,tdTomatofl/fl and Pth1rfl/fl,tdTomatofl/fl were used as controls. The skeletal phenotype was characterized by microscopic computed tomography (CT) and dynamic histomorphometry at 4, 7, and 12 weeks of age. Bone marrow adipose tissue (BMAT) was assessed by osmium tetroxide staining and CT analysis. The proximal one-third of the tibiae was designated as regulated BMAT (rBMAT) and the distal one-third was designated as constitutive BMAT (cBMAT). Colony forming unit (CFU) assays, flow cytometry, and fluorescence-activated cell sorting (FACS) were performed on flushed bone marrow stromal cells (BMSC). Two-way ANOVA and Student’s t-test were used for statistical analysis ( = 0.05). Results: Pth1rMALPs female mice exhibited a 54.2%, 15.8%, and 42.7% increase in trabecular bone volume at 4, 7, and 12 weeks of age, respectively (p = 0.001). The increase in bone volume was associated with a significant increase in labeled surfaces (MS/BS and dLS/BS, p = 0.0066 and p = 0.0429, respectively by two-way ANOVA) but an increase in bone formation rate (BFR/BS) was significant only at 4 weeks of age (p = 0.03). Flow cytometry analysis revealed that osteochondrogenic progenitor cells were decreased by half in Pth1rMALPs mice (p = 0.01). Since mineralizing surfaces were increased, these results suggest a shift of the progenitor pool towards differentiated and functional osteoblasts. Additionally, the number of CFU-F colonies was significantly decreased in Pth1rMALPs cells, further confirming the decrease in the progenitors. In contrast, the CFU-Ob remained similar between the two groups, suggesting an increase in the osteogenic potential of cells from Pth1rMALPs mice despite their decreased number. Only 6.4% ± 4.6% of CFU-Ob cells represented a distinct population of tdTomato+ cells, and 5.7% ± 2.7% had a dim tdTomato+ signal (herein, tdTomato+ dim). Interestingly, cells from Pth1rMALPs mice exhibited an 8-fold increase in tdTomato+ dim cells, and unfractionated CFU-Ob cells exhibited a 90.3% decrease in Pth1r mRNA gene expression (p = 0.001). Additionally, Pth1rMALPs mice showed a significantly higher rBMAT density, confirming that PTH suppresses adipogenesis. Importantly, sorted MALPs expressed Rankl, and its expression is increased in MALPs lacking Pth1r. The endosteal osteoclast number and surface was increased in Pth1rMALPs mice, indicating that PTH signaling in MALPs suppress osteoclastogenesis. Conclusions: Deletion of the Pth1r in MALPs partially prevents their suppression of osteogenesis through driving the progenitor pool to a differentiated state. Moreover, PTH signaling in MALPs suppresses adipogenesis and osteoclastogenesis.