An integrated understanding of the physiological response to elevated extracellular phosphate

Abstract

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.

The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

“…Previous work has indicated that P i levels influence ATP production (30). Indeed, we found that our P i starvation protocol reduced [ATP] by 66% (Fig.…”

“…8 Accumulation-Our main experimental paradigm has been to impose a nutritional challenge upon cultured cells: deprive and then restore the levels of P i found in serum. Other groups have studied the impact upon signal transduction cascades when cells are incubated with "high" [P i ] (5-10 mM) (2,30). We noted that such dramatic fluctuations in extracellular P i concentration (up to 10 mM or more) are physiologically relevant for cells that line the gastrointestinal tract (12).…”

“…PPIP5K phosphatase activities were measured at 37°C in 30-min incubations comprising 100 l of assay buffer containing 1 mM Na 2 EDTA, 50 mM KCl, 20 mM HEPES (pH 7.2), 2 mM MgCl 2 , 0.5 mg/ml BSA, and either 1 or 0.05 M chemically synthesized 1-InsP 7 (48) or InsP 8 (30). Each incubation also contained ϳ2000 dpm of the corresponding 3 H-labeled substrate.…”

“…It is also possible that regulation of PPIP5K activity by P i is not the only cell signaling mechanism that regulates InsP 8 levels, particularly those that might be activated by high extracellular [P i ] (2,30). This would not be surprising considering the many interactions between inositol pyrophosphate signaling and metabolic homeostasis (6,7,24,42) and the domain complexities of the PPIP5Ks (Fig.…”

Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake

“…In addition to the widespread structural and metabolic functions of Pi, it has become increasingly apparent during the past 15 years that extracellular Pi can act as a signaling molecule directly altering gene expression and cell phenotype (6)(7)(8)(9). The abundance of Pi in the skeleton has led to early studies describing the effects of extracellular Pi in this organ.…”