Abstract
Sustained exposure to high levels of parathyroid hormone (PTH), as observed in hyperparathyroidism, is catabolic to bone. The increase in the RANKL/OPG ratio in response to continuous PTH, resulting in increased osteoclastogenesis, is well established. However, the effects of prolonged PTH exposure on key regulators of skeletal mineralisation have yet to be investigated. This study sought to examine the temporal expression of PHOSPHO1, TNAP and nSMase2 in mineralising osteoblast-like cell cultures and to investigate the effects of continuous PTH exposure on the expression of these enzymes in vitro. PHOSPHO1, nSMase2 and TNAP expression in cultured MC3T3-C14 cells significantly increased from day 0 to day 10. PTH induced a rapid downregulation of Phospho1 and Smpd3 gene expression in MC3T3-C14 cells and cultured hemi-calvariae. Alpl was differentially regulated by PTH, displaying upregulation in cultured MC3T3-C14 cells and downregulation in hemi-calvariae. PTH was also able to abolish the stimulatory effects of bone morphogenic protein 2 (BMP-2) on Smpd3 and Phospho1 expression. The effects of PTH on Phospho1 expression were mimicked with the cAMP agonist forskolin and blocked by the PKA inhibitor PKI (5-24), highlighting a role for the cAMP/PKA pathway in this regulation. The potent down-regulation of Phospho1 and Smpd3 in osteoblasts in response to continuous PTH may provide a novel explanation for the catabolic effects on the skeleton of such an exposure. Furthermore, our findings support the hypothesis that PHOSPHO1, nSMase2 and TNAP function cooperatively in the initiation of skeletal mineralisation.
Highlights
Phosphatases are essential regulators of skeletal mineralisation, modifying local levels of inorganic phosphate (Pi), inorganic pyrophosphate (PPi) and the phosphorylation status of osteopontin, a key regulator of extracellular matrix (ECM) mineralisation [1]
It is likely that neutral sphingomyelinase 2 (nSMase2) and PHOSPHO1 function together to control the initiation of mineralisation via the liberation of Pi within the sheltered confines of osteoblast and chondrocyte matrix vesicles (MV) [1]
This possibility is supported by the observation that both PHOSPHO1 and nSMase2 are present in MV’s [27]
Summary
Phosphatases are essential regulators of skeletal mineralisation, modifying local levels of inorganic phosphate (Pi), inorganic pyrophosphate (PPi) and the phosphorylation status of osteopontin, a key regulator of extracellular matrix (ECM) mineralisation [1]. The importance of tissuenonspecific alkaline phosphatase (TNAP) as an essential component of successful skeletal mineralisation is perhaps most striking in the condition known as hypophosphatasia (HPP). Chondrocyte- and osteoblast-derived matrix vesicles (MV) from both HPP patients and Alpl-/mice retain the ability to initiate intra-vesicular mineralisation and contain hydroxyapatite crystals [3, 4], highlighting that TNAP is not essential for the initiation of MVmediated ECM mineralisation. Active within chondrocyte- and osteoblast-derived MV [5, 6] and expressed exclusively in mineralising bone, cartilage and dentin [7,8,9], PHOSPHO1, a member of the haloacid dehalogenase superfamily, is essential for the initiation of MV-mediated mineralisation. Small-molecule inhibition of PHOSPHO1 in MV’s derived from Alpl-/mice significantly affects their ability to calcify in vitro [7]
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