PTH Differentially Regulates Expression of RANKL and OPG

Abstract

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

Oxidative Stress Induces Vascular Calcification through Modulation of the Osteogenic Transcription Factor Runx2 by AKT Signaling

“…Interestingly, we found that the expression of early differentiation markers for osteoblasts ALP and Col I, was sustained even after 2 weeks in calcifying VSMC (data not shown). Such an expression pattern of ALP and Col I is different from the expression pattern of these genes during osteoblastic differentiation of bone marrow stromal cells (52). Thus, our observations indicate that oxidative stress-induced osteogenic differentiation of VSMC is not simply a mirror image of osteogenic differentiation of osteoblasts.…”

Association between Increased Serum Osteoprotegerin Levels and Improvement in Bone Mineral Density after Parathyroidectomy in Hemodialysis Patients

“…Calciotropic hormones and cytokines, including PTH, apparently act through a dual mode to inhibit OPG production and stimulate RANKL production (Vega et al 2007). Previous in vitro studies of osteoblasts have indicated that PTH either decreases (Huang et al 2004) or does not affect (Coetzee et al 2007) OPG levels. Decreased OPG gene expression was detected in bone biopsies of patients with primary hyperparathyroidism (PHPT), and was reversed after PTX (Stilgren et al 2004).…”

Deficiency of the G-protein α-Subunit Gsα in Osteoblasts Leads to Differential Effects on Trabecular and Cortical Bone

“…5-6 pairs of BGsKO mice and control littermates were used for each assay. The primers used for G s ␣ (16), collagen I (17), alkaline phosphatase (18), osteopontin (19), osteocalcin (20), RANKL (21), and osteoprotegrin (18) have been previously published. GAPDH primers were obtained from Applied Biosystems.…”