Inorganic phosphate (P) is essential for signal transduction and cell metabolism, and is also an essential structural component of the extracellular matrix of the skeleton. P is sensed in bacteria and yeast at the plasma membrane, which activates intracellular signal transduction to control the expression of P transporters and other genes that control intracellular P levels. In multicellular organisms, P homeostasis must be maintained in the organism and at the cellular level, requiring an endocrine and metabolic P-sensing mechanism, about which little is currently known. This Review will discuss the metabolic effects of P, which are mediated by P transporters, inositol pyrophosphates and SYG1-Pho81-XPR1 (SPX)-domain proteins to maintain cellular phosphate homeostasis in the musculoskeletal system. In addition, we will discuss how P is sensed by the human body to regulate the production of fibroblast growth factor 23 (FGF23), parathyroid hormone and calcitriol to maintain serum levels of P in a narrow range. New findings on the crosstalk between iron and P homeostasis in the regulation of FGF23 expression will also be outlined. Mutations in components of these metabolic and endocrine phosphate sensors result in genetic disorders of phosphate homeostasis, cardiomyopathy and familial basal ganglial calcifications, highlighting the importance of this newly emerging area of research.

Low blood phosphate (Pi) reduces muscle function in hypophosphatemic disorders. Which Pi transporters are required and whether hormonal changes due to hypophosphatemia contribute to muscle function is unknown. To address these questions we generated a series of conditional knockout mice lacking one or both housekeeping Pi transporters Pit1 and Pit2 in skeletal muscle (sm), using the postnatally expressed human skeletal actin-cre. Simultaneous conditional deletion of both transporters caused skeletal muscle atrophy, resulting in death by postnatal day P13. smPit1 −/− , smPit2 −/− and three allele mutants are fertile and have normal body weights, suggesting a high degree of redundance for the two transporters in skeletal muscle. However, these mice show a gene-dose dependent reduction in running activity also seen in another hypophosphatemic model (Hyp mice). In contrast to Hyp mice, grip strength is preserved. Further evaluation of the mechanism shows reduced ERK1/2 activation and stimulation of AMP kinase in skeletal muscle from smPit1 −/− ; smPit2 −/− mice consistent with energy-stress. Similarly, C2C12 myoblasts show a reduced oxygen consumption rate mediated by Pi transport-dependent and ERK1/2-dependent metabolic Pi sensing pathways. In conclusion, we here show that Pit1 and Pit2 are essential for normal myofiber function and survival, insights which may improve management of hypophosphatemic myopathy. Inorganic phosphate (Pi) is involved in various cellular processes including DNA and cell membrane synthesis, signal transduction, ATP production, and bone mineralization. Serum Pi is regulated by a hormonal bone-parathyroid-kidney axis consisting of fibroblast growth factor 23 (FGF23), parathyroid hormone (PTH), and 1,25(OH) 2-D (calcitriol) 1. Familial disorders of Pi homeostasis are caused by mutations in components of this axis that either directly or indirectly (via homeostatic mechanisms) lower serum Pi levels. Furthermore, chronic hypophosphatemia is observed in the often vitamin D-, and therefore Pi-, deficient elderly population 2. How muscle weakness develops in these hypophosphatemic conditions, which Pi transporters are involved and whether the homeostatic hormonal changes, which develop as a result of hypophosphatemia, contribute to reduced muscle function is poorly understood. Previous studies suggest that decreased ATP 3,4 and phosphodiesters 5 may in part explain the muscle weakness seen in hypophosphatemia. We recently reported that hypophosphatemic myopathy goes along with reduced ATP flux (V ATP) and intracellular Pi in an individual with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and in the sodium-Pi co-transporter Npt2a null mouse model of this disorder 6. Basal and insulin-stimulated muscle V ATP and Pi uptake have furthermore been shown to be decreased in the offspring of patients with type 2 diabetes 7 .

To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal.

The brain is a major sanctuary site for metastatic cancer cells that evade systemic therapies. Through pre-clinical pharmacological, biological, and molecular studies, we characterize the functional link between drug resistance and central nervous system (CNS) relapse in Epidermal Growth Factor Receptor- (EGFR-) mutant non-small cell lung cancer, which can progress in the brain when treated with the CNS-penetrant EGFR inhibitor osimertinib. Despite widespread osimertinib distribution in vivo, the brain microvascular tumor microenvironment (TME) is associated with the persistence of malignant cell sub-populations, which are poised to proliferate in the brain as osimertinib-resistant lesions over time. Cellular and molecular features of this poised state are regulated through a Ras homolog family member A (RhoA) and Serum Responsive Factor (SRF) gene expression program. RhoA potentiates the outgrowth of disseminated tumor cells on osimertinib treatment, preferentially in response to extracellular laminin and in the brain. Thus, we identify pre-existing and adaptive features of metastatic and drug-resistant cancer cells, which are enhanced by RhoA/SRF signaling and the brain TME during the evolution of osimertinib-resistant disease.

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