RANKL and OPG gene expressions were measured with and without PTH at different stages of osteoblast development. Mouse stromal cells were cultured in osteoblast differentiating conditions, and RANKL, OPG, COL1, ALP, OC, and PTHRec genes were measured using qRT-PCR. OPG:RANKL ratios indicate that PTH may induce a possible switch in the regulatory mechanism of osteoclastogenesis where OPG is inhibited early and RANKL is increased at late stages of osteoblast differentiation. Introduction: RANKL is essential for osteoclastogenesis, and its decoy receptor osteoprotegerin (OPG) negatively regulates this process. Both genes are expressed in cells of the osteoblast lineage, but the precise relationship between the state of osteoblast differentiation and RANKL and OPG expression is not clearly defined. The goal of this project was to quantify changes in RANKL and OPG gene expression in response to parathyroid hormone (PTH) at different stages of osteoblast differentiation. In this study, mouse primary bone marrow stromal cells (BMSCs) were cultured for up to 28 days. At specific time-points of cell culture, cells were stimulated with bovine PTH peptide [bPTH (1-34)] for 2 h. Levels of RANKL, OPG, ␣-1 (type I) collagen (COL1), alkaline phosphatase (ALP), osteocalcin (OC), and PTH receptor (PTHRec) mRNA were assayed using quantitative real-time reverse-transcriptasepolymerase chain reaction (qRT-PCR). Materials and Methods: In control cells, there was a gradual increase of RANKL gene expression with murine osteoblastic stromal cell maturation to a 3-fold level at day 28. In contrast, OPG mRNA levels were maximal at day 14 of cell culture and decreased through the latter stages of osteoblast differentiation. Exposing the cells to 100 ng/ml of bPTH(1-34) induced minimal increases in RANKL mRNA levels from days 7 to 14 but elevated expression significantly at days 21 (2-fold) and 28 (3-fold). PTH inhibited OPG gene expression maximally at day 14, but continued to have inhibitory effects on cultured cells at days 21 and 28. Alterations of RANKL and OPG mRNA levels by PTH in day 14 osteoblasts were sufficient to sustain a 5.6-fold increase in the number of TRACP ϩ cells when cocultured with osteoclast precursor cells. Cells in culture after 28 days showed a 1.9-fold increase in TRACP

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In some G protein-coupled receptors (GPCRs), agonist-dependent phosphorylation by specific GPCR kinases (GRKs) is an important mediator of receptor desensitization and endocytosis. Phosphorylation and the subsequent events that it triggers, such as arrestin binding, have been suggested to be regulatory mechanisms for a wide variety of GPCRs. In the present study, we investigated whether agonist-induced phosphorylation of the PTH receptor, a class II GPCR, also regulates receptor internalization. Upon agonist stimulation, the PTH receptor was exclusively phosphorylated on serine residues. Phosphoamino acid analysis of a number of receptor mutants in which individual serine residues had been replaced by threonine identified serine residues in positions 485, 486, and 489 of the cytoplasmic tail as sites of phosphorylation after agonist treatment. When serine residues at positions 483, 485, 486, 489, 495, and 498 were simultaneously replaced by alanine residues, the PTH receptor was no longer phosphorylated either basally or in response to PTH. The substitution of these serine residues by alanine affected neither the number of receptors expressed on the cell surface nor the ability of the receptor to signal via Gs. Overexpression of GRK2, but not GRK3, enhanced PTH-stimulated receptor phosphorylation, and this phosphorylation was abolished by alanine mutagenesis of residues 483, 485, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor by the endogenous kinase in HEK-293 cells occurs on the same residues targeted by overexpressed GRK2. Strikingly, the rate and extent of PTH-stimulated internalization of mutated PTH receptors lacking phosphorylation sites were identical to that observed for the wild-type PTH receptor. Moreover, overexpressed GRK2, while enhancing the phosphorylation of the wild-type PTH receptor, had no affect on the rate or extent of receptor internalization in response to PTH. Thus, the agonist-occupied PTH receptor is phosphorylated by a kinase similar or identical to GRK2 in HEK-293 cells, but this phosphorylation is not requisite for efficient receptor endocytosis.

G protein-coupled receptors (GPCRs) coupled to activation of Gs, such as the PTH1 receptor (PTH1R), have long been known to regulate skeletal function and homeostasis. However, the role of GPCRs coupled to other G proteins such as Gi is not well established. We used the tet-off system to regulate the expression of an activated Gi-coupled GPCR (Ro1) in osteoblasts in vivo. Skeletal phenotypes were assessed in mice expressing Ro1 from conception, from late stages of embryogenesis, and after weaning. Long bones were assessed histologically and by microcomputed tomography. Expression of Ro1 from conception resulted in neonatal lethality that was associated with reduced bone mineralization. Expression of Ro1 starting at late embryogenesis resulted in a severe trabecular bone deficit at 12 wk of age (>51% reduction in trabecular bone volume fraction in the proximal tibia compared with sex-matched control littermates; n = 11; P < 0.01). Ro1 expression for 8 wk beginning at 4 wk of age resulted in a more than 20% reduction in trabecular bone volume fraction compared with sex-matched control littermates (n = 16; P < 0.01). Bone histomorphometry revealed that Ro1 expression is associated with reduced rates of bone formation and mineral apposition without a significant change in osteoblast or osteoclast surface. Our results indicate that signaling by a Gi-coupled GPCR in osteoblasts leads to osteopenia resulting from a reduction in trabecular bone formation. The severity of the phenotype is related to the timing and duration of Ro1 expression during growth and development. The skeletal phenotype in Ro1 mice bears some similarity to that produced by knockout of Gs-alpha expression in osteoblasts and thus may be due at least in part to Gi-mediated inhibition of adenylyl cyclase.