The Role of Sphingosine Kinases in Bone Anabolism

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Project Summary/Abstract Parathyroid hormone is a major systemic regulator of the basal rate of bone formation, which is why patients with hypoparathyroidism have very low rates of bone formation, while bone formation rates are increased in hyperparathyroidism. The ability of PTH to stimulate bone formation is the basis for its use as the only FDA- approved anabolic agent (teriparatide) to treat low bone mass. CSF-1 is the principle colony stimulating activity released by osteoblasts constitutively and in response to PTH treatment, and its receptor, c-fms, is more highly expressed on osteoclasts than any other cell in bone or in the hematopoietic microenvironment. We have identified several key actions of CSF-1 on the formation and function of mature osteoclasts. We also have very recently discovered that CSF-1 transcriptionally activates the sphingosine kinase 1 gene (SPHK1) in mature osteoclasts, which is the rate-limiting enzyme required for the synthesis of sphingosine-1- phosphate (S-1-P), a phospholipid with anabolic effects in bone. This change results in an increase in S-1-P production by osteoclasts. Two recently reported mouse models in which a selective increase in osteoclast production of S-1-P results in an increased basal rate of bone formation in vivo and an increase in bone mass support the physiologic importance of this phospholipid as a key regulator of bone anabolism. Together with our data, these findings lead us to propose a new endocrine/paracrine loop in which PTH stimulates the production of CSF-1 from cells in the osteoblast lineage resulting in increased expression of SPHK1 and S-1-P production by osteoclasts. This locally produced S-1-P in bone “feeds back” on osteoblasts to contribute to the bone forming actions of PTH. Based on this model we hypothesize that SPHK1 and S-1-P are part of the downstream anabolic path entrained by single daily administration of PTH. To test this hypothesis we will undertake the following specific aims: 1. Directly determine the contribution of sphingosine kinases to the basal rate of bone formation by selectively deleting SPHK activity in osteoclasts in vivo to determine its contribution to bone anabolism as quantified by changes in bone mass and bone formation rates as well as changes in the function of osteoblasts isolated from SPHK null mice. 2. Determine the contribution of sphingosine kinases to the anabolic response to PTH by examining the anabolic response to single daily administration of PTH to SPHK null mice. If the anabolic response is attenuated we will undertake gene-profiling studies using PCR-expression arrays that target bone anabolism to begin to understand the molecular bases for the observed differences. 3. Examine how CSF-1 increases expression of sphingosine kinase 1 (SPHK1) by studying the mechanisms by which of CSF-1 transcriptionally activates the SPHK1 promoter and determining if blocking ERK signaling prevents this. We will also evaluate the effect of CSF-1 on the half-life of SPHK1 mRNA.