Vascular calcification (VC) is the inappropriate deposition of calcium phosphate mineral in the vasculature, and arterial calcification can cause cardiovascular complications through hypertension, decreased vascular compliance, and increased pulse wave velocity. VC occurs in the normal ageing population, but patients with chronic kidney disease (CKD) have a highly increased prevalence of VC compared to age- matched controls. In CKD patients, reduced renal function diminishes the ability to excrete inorganic phosphate (Pi), which leads to hyperphosphatemia. Elevated Pi is a recognized risk factor for cardiovascular morbidity and mortality in the CKD population due to increased calcification, which is caused by active deposition of mineral by vascular smooth muscle cells (VSMCs). Elevated Pi induces VSMCs to undergo an osteochondrogenic phenotype transition, which involves a decrease in SMC markers (SM22a, SMa-actin, SM- MHC) and an increase in osteochondrogenic markers (Runx2, OPN, OCN, ALP). However, the mechanism of Pi-induced VSMC phenotype change and matrix mineralization is unclear. Our lab has previously shown that the type III sodium-dependent phosphate co-transporter, PiT-1, is the main Pi transporter in human VSMCs and is required for human VSMC matrix mineralization and osteochondrogenic differentiation in vitro. Although this suggested PiT-1 promoted mineralization through increased Pi uptake, recent studies have questioned that conclusion. Pi uptake through PiT-1 was shown to be saturated at Pi concentrations below 0.5 mM, which is well below the Pi concentration required to induce mineralization (around 2.4 mM), suggesting Pi uptake is not required for Pi-induced effects. Recently, our lab has observed that elevated Pi (3.0 mM) induces ERK1/2 phosphorylation in VSMCs, and deletion of PiT-1 from VSMCs removed this induction. Furthermore, we could rescue Pi-induced ERK1/2 phosphorylation and osteochondrogenic differentiation of VSMCs by overexpression of either wild-type PiT-1 or a Pi-deficient PiT-1 mutant protein. These results suggest PiT-1 can sense and respond to elevated Pi by a Pi uptake-independent mechanism through ERK1/2 cell signaling. Given preliminary data and previous studies, we hypothesize that elevated Pi induces PiT-1 transition from a dimer to a monomer state, which exposes a cryptic site and allows for protein interactions with the receptor signal initiator protein RAPGEF1 and causes activation of the RAF/MEK/ERK signaling pathway. In this proposal, we aim to elucidate the mechanisms of PiT-1 cell signaling by 1) investigating the importance of RAPGEF1 in Pi-induced ERK1/2 phosphorylation and protein binding between RAPGEF1 and PiT-1 in VSMCs, and 2) investigating the role of PiT-1 dimerization in response to elevated Pi and the functional PiT-1 domains required for dimerization. This novel signaling pathway is the first Pi sensing pathway discovered in VSMCs. We believe that this pathway can provide novel therapeutic strategies that could inhibit the effects of Pi on VSMCs and block hyperphosphatemia-induced VC in CKD patients.
Vascular calcification increases the risk of cardiovascular morbidity and mortality, and inorganic phosphate promotes vascular calcification by causing vascular smooth muscle cells to deposit calcium phosphate mineral. We will study the main phosphate transporter, PiT-1, and its role in promoting vascular smooth muscle cell mineralization. A better understanding of how phosphate causes calcification could lead to therapies that block calcification, which are currently unavailable.
