Person:

Bergwitz, Clemens

Maintenance of physiologic phosphate balance is of crucial biological importance, as it is fundamental to cellular function, energy metabolism, and skeletal mineralization. Fibroblast growth factor-23 (FGF-23) is a master regulator of phosphate homeostasis, but the molecular mechanism of such regulation is not yet completely understood. Targeted disruption of the Fgf-23 gene in mice (Fgf-23[super]−/−) elicits hyperphosphatemia, and an increase in renal sodium/phosphate co-transporter 2a (NaPi2a) protein abundance. To elucidate the pathophysiological role of augmented renal proximal tubular expression of NaPi2a in Fgf-23[super]−/− mice and to examine serum phosphate–independent functions of Fgf23 in bone, we generated a new mouse line deficient in both Fgf-23 and NaPi2a genes, and determined the effect of genomic ablation of NaPi2a from Fgf-23[super]−/− mice on phosphate homeostasis and skeletal mineralization. Fgf-23[super]−/−/NaPi2a[super]−/− double mutant mice are viable and exhibit normal physical activities when compared to Fgf-23[super]−/− animals. Biochemical analyses show that ablation of NaPi2a from Fgf-23[super]−/− mice reversed hyperphosphatemia to hypophosphatemia by 6 weeks of age. Surprisingly, despite the complete reversal of serum phosphate levels in Fgf-23[super]−/−/NaPi2a[super]−/−, their skeletal phenotype still resembles the one of Fgf23[super]−/− animals. The results of this study provide the first genetic evidence of an in vivo pathologic role of NaPi2a in regulating abnormal phosphate homeostasis in Fgf-23[super]−/− mice by deletion of both NaPi2a and Fgf-23 genes in the same animal. The persistence of the skeletal anomalies in double mutants suggests that Fgf-23 affects bone mineralization independently of systemic phosphate homeostasis. Finally, our data support (1) that regulation of phosphate homeostasis is a systemic effect of Fgf-23, while (2) skeletal mineralization and chondrocyte differentiation appear to be effects of Fgf-23 that are independent of phosphate homeostasis.

Phosphate is required for many important cellular processes and having too little phosphate or too much can cause disease and reduce life span in humans. However, the mechanisms underlying homeostatic control of extracellular phosphate levels and cellular effects of phosphate are poorly understood. Here, we establish Drosophila melanogaster as a model system for the study of phosphate effects. We found that Drosophila larval development depends on the availability of phosphate in the medium. Conversely, life span is reduced when adult flies are cultured on high phosphate medium or when hemolymph phosphate is increased in flies with impaired Malpighian tubules. In addition, RNAi-mediated inhibition of MAPK-signaling by knockdown of Ras85D, phl/D-Raf or Dsor1/MEK affects larval development, adult life span and hemolymph phosphate, suggesting that some in vivo effects involve activation of this signaling pathway by phosphate. To identify novel genetic determinants of phosphate responses, we used Drosophila hemocyte-like cultured cells (S2R+) to perform a genome-wide RNAi screen using MAPK activation as the readout. We identified a number of candidate genes potentially important for the cellular response to phosphate. Evaluation of 51 genes in live flies revealed some that affect larval development, adult life span and hemolymph phosphate levels.

The major facilitator superfamily (MFS) transporter (Pho84) and the type III transporter (Pho84) are responsible for metabolic effects of inorganic phosphate in yeast. While the (Pho84) ortholog Pit1 was also shown to be involved in phosphate-activated MAPK in mammalian cells, it is currently unknown, whether orthologs of (Pho84) have a role in phosphate-sensing in metazoan species. We show here that the activation of MAPK by phosphate observed in mammals is conserved in (Drosophila) cells, and used this assay to characterize the roles of putative phosphate transporters. Surprisingly, while we found that RNAi-mediated knockdown of the fly (Pho84) ortholog dPit had little effect on the activation of MAPK in (Drosophila) S2R+ cells by phosphate, two (Pho84)/SLC17A1–9 MFS orthologs (MFS10 and MFS13) specifically inhibited this response. Further, using a (Xenopus) oocyte assay, we show that MSF13 mediates uptake of [33P]-orthophosphate in a sodium-dependent fashion. Consistent with a role in phosphate physiology, MSF13 is expressed highest in the (Drosophila) crop, midgut, Malpighian tubule, and hindgut. Altogether, our findings provide the first evidence that (Pho84) orthologs mediate cellular effects of phosphate in metazoan cells. Finally, while phosphate is essential for (Drosophila) larval development, loss of MFS13 activity is compatible with viability indicating redundancy at the levels of the transporters.