Phosphate uptake-independent signaling functions of the type III sodium-dependent phosphate transporter, PiT-1, in vascular smooth muscle cells

Abstract

Vascular calcification (VC) is prevalent in chronic kidney disease and elevated serum inorganic phosphate (Pi) is a recognized risk factor. The type III sodium-dependent phosphate transporter, PiT-1, is required for elevated Pi-induced osteochondrogenic differentiation and matrix mineralization in vascular smooth muscle cells (VSMCs). However, the molecular mechanism(s) by which PiT-1 promotes these processes is unclear. In the present study, we confirmed that the Pi concentration required to induce osteochondrogenic differentiation and matrix mineralization of mouse VSMCs was well above that required for maximal Pi uptake, suggesting a signaling function of PiT-1 that was independent of Pi transport. Elevated Pi-induced signaling via ERK1/2 phosphorylation was abrogated in PiT-1 deficient VSMCs, but could be rescued by wild-type (WT) and a Pi transport-deficient PiT-1 mutant. Furthermore, both WT and transport-deficient PiT-1 mutants promoted osteochondrogenic differentiation as measured by decreased SM22α and increased osteopontin mRNA expression. Finally, compared to vector alone, expression of transport-deficient PiT-1 mutants promoted VSMC matrix mineralization, but not to the extent observed with PiT-1 WT. These data suggest that both Pi uptake-dependent and -independent functions of PiT-1 are important for VSMC processes mediating vascular calcification.

Keywords: Calcification; ERK phosphorylation; Phosphate; PiT-1; SLC20A1; Signaling.

Figure 1. Concentrations of Pi above levels that support maximal Pi uptake rate are required to induce matrix mineralization and osteochondrogenic differentiation in VSMC

(A) Sodium-dependent Pi uptake was measured at different Pi concentrations (0.03 mM to 0.5 mM Pi) in VSMCs isolated from wild-type C57BL/6 mice (WT VSMCs). (B) Calcium deposition of WT VSMCs was quantified after 8 days of incubated in various Pi concentrations. (C–D) Separately, RNA lysate of WT VSMCs was collected after 6 days of Pi induction, and then (C) OPN and (D) SM22α were quantified by Q-PCR. Data are presented as mean ± standard deviation (S.D.), n = 3 for all data points. Statistically significant differences from 1.0 mM Pi data (B–D) are indicated by * = P<0.05 as measured by One-way ANOVA post-hoc Tukey analysis.

Figure 2. PiT-1 is required for Pi-induced ERK1/2 phosphorylation in VSMCs

(A) Pi induction of p-ERK1/2 and total ERK1/2 were visualized by Western blot analysis by incubating PiT-1 fl/fl and PiT-1 ΔSM VSMCs in 0.5 mM Pi, 1.0 mM Pi, 3.0 mM Pi, or 3.0 mM Sodium sulfate for 5 or 15 minutes. (B) Densitometry was used to quantify the immunoblot images and shown as the ratio of P-ERK1/2 to Total ERK1/2. Western blot is representative of three experiments with similar results, and quantification is represented as mean ± S.D., n = 3 for all data points. Statistically significant differences from all other data from the same cell line is indicated by * = P<0.05 as measured by One-way ANOVA post-hoc Tukey analysis.

Figure 3. PiT-1 constructs transduced into PiT-1 ΔSM VSMCs are expressed and did not alter PiT-2 mRNA levels

(A) Q-PCR quantification of PiT-1 mRNA expression of PiT-1 ΔSM VSMCs transduced with vector control, PiT-1 WT, PiT-1-E74K, PiT-1-S132A, or PiT-1-S623A confirmed stable expression. (B) Immunocytochemical analysis of PiT-1 ΔSM VSMCs expressing Vector Control, PiT-1-WT, PiT-1-E74K, PiT-1-S132A, or PiT-1-S623A with primary PiT-1 antibody (red) and DAPI counterstain (blue) show expression of the PiT-1 WT, PiT-1-E74K, PiT-1-S132A, and PiT-1-S623A proteins, scale bar is 25 µm. (C) PiT-2 mRNA quantification by Q-PCR confirmed no significant effects on PiT-2 mRNA expression in any cell line. Data presented as mean ± S.D. (A,C) or representative images (B).

Figure 4. PiT-1 point mutations impair sodium-dependent Pi uptake in VSMCs

Sodium-dependent Pi uptake was quantified over a range of Pi concentrations in VSMCs expressing vector control, PiT-1 WT, PiT-1-E74K, PiT-1-S132A, or PiT-1-S623A. VSMC Pi uptake was measured over 20 minutes and normalized to time and VSMC protein content. Dashed connecting lines signify PiT-1 point mutation constructs. Data presented as mean ± S.D., n = 3 for all data points. Statistically significant differences between Vector Control and each PiT-1 construct at the same Pi concentration are indicated by * = P<0.05 as measured by One-way ANOVA post-hoc Tukey analysis.

Figure 5. Elevated Pi induced ERK1/2 phosphorylation through Pi transport-independent PiT-1 function

(A) ERK1/2 phosphorylation was induced in PiT-1 ΔSM VSMCs expressing vector control, PiT-1 WT, or PiT-1-E74K with incubation in 1.0 mM or 3.0 mM Pi for 15 minutes. P-ERK1/2 and total ERK1/2 were visualized by western blot analysis. (B) Densitometry quantification of three independent experiments shows the ratio of P-ERK1/2 to Total ERK1/2. Data presented as a representative image (A) or mean ± S.D., n = 3 for all data points (B). Statistically significant differences between two independent means are indicated by * = P<0.05 as measured by student t-test.

Figure 6. PiT-1 promotion of VSMC osteochondrogenic differentiation does not require Pi uptake

(A) SM22α RNA expression in PiT-1 ΔSM VSMCs expressing vector control, PiT-1 WT, or PiT-1-E74K was quantified by Q-PCR after incubation in 1.0 mM Pi for 4 days. (B) OPN RNA expression is presented as fold-induction of 2.6 mM Pi over 1.0 mM Pi after 4 days of incubation. Data presented as mean ± S.D., n = 3 for all data points. Statistically significant differences of means compared to vector control are indicated by * = P<0.05 as measured by One-way ANOVA post-hoc Tukey analysis.

Figure 7. PiT-1 promotes VSMC matrix mineralization through both Pi uptake-dependent and -independent functions

(A) Matrix calcium content was quantified from PiT-1 ΔSM VSMCs expressing vector control, PiT-1 WT, or PiT-1-E74K that were induced to mineralize in normal Pi (1.0 mM) or elevated Pi (2.6 mM) for 8 days. (B) Rate of apoptosis was determined after incubation in normal or elevated Pi for 2 or 5 days. (C) Calcification was quantified of PiT-1 ΔSM VSMCs expressing vector control, PiT-1 WT, PiT-1-E74K, PiT-1-S132A, or PiT-1-S623A, and data is presented as fold-induction over vector control for each experiment by cell line. (D) Correlation analysis between calcification and calculated sodium-dependent Pi uptake Vmax parameter for each cell line is presented, with a horizontal dash indicating average value of data points and a dashed regression line with the coefficient of correlation indicating linear correlation. Data is presented as mean ± S.D. (A–C), or as single points for linear regression (D). Statistically significant differences between indicated means (A) or compared to vector control (C) are indicated by * = P<0.05, determined by One-way ANOVA post-hoc Tukey analysis.