Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi-phase, extracellular phosphate-induced, signaling network in pre-osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c-fos. The mechanism of activation requires FGF receptor signaling followed by stimulation of N-Ras and activation of AP-1 and serum response elements. A distinct long-term response also requires FGF receptor signaling and results in N-Ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long-term cell behavior.

Thiazolidinedione (TZD) therapy has been associated with an increased risk of bone fractures. Studies in rodents have led to a model in which decreased bone quality in response to TZDs is due to a competition of lineage commitment between osteoblasts (OBs) and adipocytes (ADs) for a common precursor cell, resulting in decreased OB numbers. Our goal was to investigate the effects of TZD exposure on OB-AD lineage determination from primary human bone marrow stromal cells (hBMSCs) both in vitro and in vivo from nondiabetic subjects and patients with type 2 diabetics. Our experimental design included 2 phases. Phase 1 was an in vitro study of TZD effects on the differentiation of hBMSCs into OBs and ADs in nondiabetic subjects. Phase 2 was a randomized, placebo-controlled trial to determine the effects of 6-month pioglitazone treatment in vivo on hBMSC differentiation using AD/OB colony forming unit assays in patients with type 2 diabetes. In vitro, TZDs (pioglitazone and rosiglitazone) enhanced the adipogenesis of hBMSCs, whereas neither altered OB differentiation or function as measured by alkaline phosphatase activity, gene expression, and mineralization. The ability of TZDs to enhance adipogenesis occurred at a specific time/stage of the differentiation process, and pretreating with TZDs did not further enhance adipogenesis. In vivo, 6-month TZD treatment decreased OB precursors, increased AD precursors, and increased total colony number in patients with type 2 diabetes. Our results indicate that TZD exposure in vitro potently stimulates adipogenesis but does not directly alter OB differentiation/mineralization or lineage commitment from hBMSCs. However, TZD treatment in type 2 diabetic patients results in decreased osteoblastogenesis from hBMSCs compared with placebo, indicating an indirect negative effect on OBs and suggesting an alternative model by which TZDs might negatively regulate bone quality.