Abstract
Parathyroid Hormone (PTH) can exert both anabolic and catabolic effects on the skeleton, potentially through expression of the PTH type1 receptor (PTH1R), which is highly expressed in osteocytes. To determine the cellular and molecular mechanisms responsible, we examined the effects of PTH on osteoblast to osteocyte differentiation using primary osteocytes and the IDG-SW3 murine cell line, which differentiate from osteoblast to osteocyte-like cells in vitro and express GFP under control of the dentin matrix 1 (Dmp1) promoter. PTH treatment resulted in an increase in some osteoblast and early osteocyte markers and a decrease in mature osteocyte marker expression. The gene expression profile of PTH-treated Day 28 IDG-SW3 cells was similar to PTH treated primary osteocytes. PTH treatment induced striking changes in the morphology of the Dmp1-GFP positive cells in IDG-SW3 cultures and primary cells from Dmp1-GFP transgenic mice. The cells changed from a more dendritic to an elongated morphology and showed increased cell motility. E11/gp38 has been shown to be important for cell migration, however, deletion of the E11/gp38/podoplanin gene had no effect on PTH-induced motility. The effects of PTH on motility were reproduced using cAMP, but not with protein kinase A (PKA), exchange proteins activated by cAMP (Epac), protein kinase C (PKC) or phosphatidylinositol-4,5-bisphosphonate 3-kinase (Pi3K) agonists nor were they blocked by their antagonists. However, the effects of PTH were mediated through calcium signaling, specifically through L-type channels normally expressed in osteoblasts but decreased in osteocytes. PTH was shown to increase expression of this channel, but decrease the T-type channel that is normally more highly expressed in osteocytes. Inhibition of L-type calcium channel activity attenuated the effects of PTH on cell morphology and motility but did not prevent the downregulation of mature osteocyte marker expression. Taken together, these results show that PTH induces loss of the mature osteocyte phenotype and promotes the motility of these cells. These two effects are mediated through different mechanisms. The loss of phenotype effect is independent and the cell motility effect is dependent on calcium signaling.
Highlights
Osteocytes are the most abundant and long lived cells within the bone and are known to play important roles in regulating bone formation, resorption and homeostasis
To determine the effects of parathyroid hormone (PTH) on osteoblast and osteocyte gene expression, IDG-SW3 cells were treated at day 0, 7, 14, 21 and 28 with 50nM PTH for 24 hours and gene expression was examined by RT-PCR
The early osteocyte marker E11/gp38/pdpn was highly expressed in the early stages of differentiation and its expression decreased in the mature cultures (Fig 1A)
Summary
Osteocytes are the most abundant and long lived cells within the bone and are known to play important roles in regulating bone formation, resorption and homeostasis. They represent the terminal differentiation stage of the osteoblast lineage, where an osteoblast has become entrapped within the mineralized matrix. Recent studies have indicated the importance of osteocytes in maintaining bone mass They are important regulators of osteoclast formation and activity [2,3,4,5] and may be the primary source of receptor activator of nuclear factor kappa-B ligand within the adult skeleton [3,4]. One of the most important and well known of these signals is parathyroid hormone (PTH), which is secreted by the parathyroid gland and is known to have both anabolic and catabolic effects on the skeleton [12]
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