Effects of PTH on RANK-L and OPG Gene Expression During Osteoblast Differentiation

Objectives: 1) Establish a correlation between osteoblast differentiation state and RANK-L / OPG gene expression, and 2) Evaluate upregulation / downregulation of RANK-L and OPG gene expression using parathyroid hormone (PTH).

Methods: Mouse bone marrow stromal cells isolated from 3-month old male C57Bl/6 mice were cultured for 28 days in the presence of ascorbic acid and beta-glycerophosphate. At Days 7, 14, 21, and 28, the cells were acutely stimulated with b(1-34) PTH (100 ng/ml) for 2 hrs. RANK-L, OPG, alpha-1 collagen, alkaline phosphatase (early bone markers), osteocalcin (late bone marker), and PTH-Receptor genes were assayed using quantitative real-time RT-PCR.

Results: Bone marker data validated that the freshly isolated stromal cells followed a predictable pattern of differentiation, with alpha-1 col and Alk. PO₄ expression peaking at 14 day. OC and PTH-Rec were maximally expressed after 21 & 28 days in culture. Acute treatment with PTH did not affect the bone marker genes. RANK-L increased as differentiation occurred and reached maximum at Day 28. OPG reached maximal expression at Day 14 (2.5-fold over Day 7 levels) and tapered off. PTH increased RANK-L expression and decreased OPG production. Alterations in RANK-L and OPG levels by PTH were sufficient to sustain a three-fold elevation in biologic osteoclastogenic potential as demonstrated by higher numbers of co-cultured TRAP+ staining colonies.

Conclusions: 1) PTH has a significant upregulation of RANK-L gene expression with maximal sensitivity of the RANK-L gene to PTH occurring relatively late in the process of osteoblast differentiation; 2) PTH has an inhibitory effect on OPG, and 3) changes in RANK-L and OPG levels following PTH exposure resulted in increased osteoclast induction potential.