Inorganic phosphate (Pi) is an essential nutrient for human health. Due to the changes in our dietary pattern, dietary Pi overload engenders systemic phosphotoxicity, including excessive Pi-related vascular calcification and chronic tissue injury. The molecular mechanisms of the seemingly distinct phenotypes remain elusive. In this study, we investigated Pi-mediated cellular response in HEK293 and HeLa cells. We found that abnormally high Pi directly mediates diverse cellular toxicity in a dosedependent manner. Up to 10 mM extracellular Pi promotes cell proliferation by activating AKT signaling cascades and augmenting cell cycle progression. By introducing additional Pi, higher than the concentration of 40 mM, we observed significant cell damage caused by the interwoven Pi-related biological processes. Elevated Pi activates mitogen-activated protein kinase (MAPK) signaling, encompassing extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and Jun amino-terminal kinase (JNK), which consequently potentiates Pi triggered lethal epithelial-mesenchymal transition (EMT). Synergistically, high Pi-caused endoplasmic reticulum (ER) stress also contributes to apparent apoptosis. To counteract, Pi-activated AKT signaling promotes cell survival by activating the mammalian target of rapamycin (mTOR) signaling and blocking ER stress. Pharmacologically or genetically abrogating Pi transport, the impact of high Pi-induced cytotoxicity could be reduced. Taken together, abnormally high extracellular Pi results in a broad spectrum of toxicity by rewiring complicated signaling networks that control cell growth, cell death, and homeostasis.

21Inorganic phosphate (Pi) is an essential nutrient for human health. Due to our change in dietary 22 pattern, dietary Pi overload engenders systematic phosphotoxicity, including excessive Pi related 23 vascular calcification and chronic tissue injury. The molecular mechanisms of the seemingly 24 distinct phenotypes remain elusive. In this study, we found that Pi directly mediates diverse 25 cellular toxicity in a dose-dependent manner on a cell-based model. At moderately higher than 26 physiological level, extracellular Pi promotes cell proliferation by activating AKT and 27 extracellular signal-regulated kinase 1/2 (ERK1/2) cascades. By introducing additional Pi, we 28 observed significant cell damage caused by the interwoven Pi related biological processes, 29 including activation of mitogen-activated protein kinase (MAPK) signaling, endoplasmic 30 reticulum (ER) stress, epithelial-mesenchymal transition (EMT) and apoptosis. Taken together, 31 elevated extracellular Pi results in a broad spectrum of toxicity by rewiring complicated 32 signaling networks that control cell growth, cell death, ER stress, and cell mobility.33 34 42also critical for skeletal and dentin formation, growth and maintenance (Camalier, Yi et al., 2013). 43 Page 3 of 35 Humans routinely consume phosphorus through food. Phosphorus is oxidized and transformed into PO 4 44 after consumption within the body. Insufficient intake of dietary phosphorus, mostly due to malnutrition, 45 can cause deficiency in skeletal mineralization, and development of rickets. In contrast, Pi overload 46 accounts for the imbalance of phosphate metabolism leading to various human health problems, such as 47 157