Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor

Abstract

It is now widely recognized that purinergic signaling plays an important role in the regulation of bone remodeling. One receptor subtype, which has been suggested to be involved in this regulation, is the P2Y2 receptor (P2Y2R). In the present study, we investigated the effect of P2Y2R overexpression on bone status and bone cell function using a transgenic rat. Three-month-old female transgenic Sprague Dawley rats overexpressing P2Y2R (P2Y2R-Tg) showed higher bone strength of the femoral neck. Histomorphometry showed increase in resorptive surfaces and reduction in mineralizing surfaces. Both mineral apposition rate and thickness of the endocortical osteoid layer were higher in the P2Y2R-Tg rats. μCT analysis showed reduced trabecular thickness and structural model index in P2Y2R-Tg rats. Femoral length was increased in the P2Y2R-Tg rats compared to Wt rats. In vitro, there was an increased formation of osteoclasts, but no change in total resorption in cultures from P2Y2R-Tg rats. The formation of mineralized nodules was significantly reduced in the osteoblastic cultures from P2Y2R-Tg rats. In conclusion, our study suggests that P2Y2R is involved in regulation of bone turnover, due to the effects on both osteoblasts and osteoclasts and that these effects might be relevant in the regulation of bone growth.

Keywords: Cell/tissue signaling; Genetic animal models; Osteoblasts; Osteoclasts; P2Y2 purinergic receptors.

Fig. 1

Micrograph of osteoid formation determined on Goldner’s trichrome-stained bone sections of the cortical bone of the tibia from P2Y2R-Tg rats and Wt rats (white arrow: bone; yellow arrow: osteoid)

Fig. 2

a Fold change in expression of P2Y2 mRNA in brain and lung tissue (n = 3) as well as calvarial osteoblasts (n = 4) of P2Y2R-Tg rats relative to Wt (n = 3 (brain and lung); n = 6 (calvarial osteoblasts)). b P2Y2R protein expression in calvarial osteoblasts from WT (n = 4) and P2Y2R-Tg (n = 4) rats as measured by Western blotting. All bands are normalized to histone H3 expression, and mean expression in Wt cells is set at 100%. Lower panel shows membrane images of each individual experiment. Error bars represent SEM. TG P2Y2R transgenic, Wt wild-type

Fig. 3

Mean change in background subtracted Fluo-4 fluorescence ± SEM as a measure of intracellular Ca2+ release for both Wt and P2Y2R-Tg rat calvarial osteoblasts (n = 5) using UTP as agonist. *p < 0.05

Fig. 4

a The total number of osteoclasts identified as TRAP-positive cells with three or more nuclei, developed from bone marrow from P2Y2R-Tg or Wt rats in ten randomly selected fields of view. b Resorption of bone slices by primary osteoclasts from P2Y2R-Tg or Wt rats (percentage of total area). Data are presented as mean ± SEM and p value from t test (n = 10)

Fig. 5

a Bone nodule formation in osteoblast cultures stained with Alizarin Red (AR-S). b Production of fibrillary collagen in osteoblast cultures measured by Sirius Red stain. c Alkaline phosphatase (ALP) activity measured as percentage of total protein content in cell lysates. AR-S data are presented as individual experiments (mean of four replicates), mean (—) and p value from Mann-Whitney U test (n = 4). Collagen and ALP data are presented as mean ± SEM and p value from t test (n = 10)