BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells

Abstract

Vascular calcification is associated with increased risk of cardiovascular events that are the most common cause of death in patients with end-stage renal disease. Clinical and experimental studies indicate that hyperphosphatemia is a risk factor for vascular calcification and cardiovascular mortality in these patients. Our previous studies demonstrated that phosphate transport through the type III sodium-dependent phosphate cotransporter, Pit-1, was necessary for phosphate-induced calcification and osteochondrogenic phenotypic change in cultured human smooth muscle cells (SMC). BMP-2 is a potent osteogenic protein required for osteoblast differentiation and bone formation that has been implicated in vascular calcification. In the present study, we have examined the effects of BMP-2 on human SMC calcification in vitro. We found that treatment of SMC with BMP-2 enhanced elevated phosphate-induced calcification, but did not induce calcification under normal phosphate conditions. mRNAs for BMP receptors, including ALK2, ALK3, ALK6, BMPR-II, ActR-IIA and ActR-IIB were all detected in human SMCs. Mechanistically, BMP-2 dose-dependently stimulated phosphate uptake in SMC (200 ng/ml BMP-2 vs. vehicle: 13.94 vs. 7.09 nmol/30 min/mg protein, respectively). Real-time PCR and Western blot revealed the upregulation of Pit-1 mRNA and protein levels, respectively, by BMP-2. More importantly, inhibition of phosphate uptake by a competitive inhibitor of sodium-dependent phosphate cotransport, phosphonoformic acid, abrogated BMP-2-induced calcification. These results indicate that phosphate transport via Pit-1 is crucial in BMP-2-regulated SMC calcification. In addition, BMP-2-induced Runx2 and inhibited SM22 expression, indicating that it promotes osteogenic phenotype transition in these cells. Thus, BMP-2 may promote vascular calcification via increased phosphate uptake and induction of osteogenic phenotype modulation in SMC.

Fig. 1

Expression of BMP receptors in human SMC. Total RNA was isolated from human immortalized SMC and cDNA synthesis was performed using Omniscript Reverse Transcriptase (Qiagen). Identical amounts of cDNA were used for PCR reactions with primers specific to ALK2, ALK3, ALK6, BMPR-II, ActRII-A and ActRII-B.

Fig. 2

BMP-2 enhances phosphate-induced SMC calcification. Confluent human immortalized SMC were incubated with various concentrations of phosphate in the presence or absence of 200 ng/ml of human recombinant BMP-2 for 10 days (A) or 2.2 mM phosphate in the presence or absence of 200 ng/ml of human recombinant BMP-2 for the indicated days (B). Calcium content of the extracellular matrix is given as mean ± S.D. (n = 3). *P < 0.05.

Fig. 3

BMP-2 increases phosphate uptake in human immortalized SMC. (A). Human SMC were seeded in 24-well plates 1 day prior to the uptake assay. After treatment with different concentrations of BMP-2 for 6 hrs, phosphate uptake assays were performed by incubation of SMC with EBSS containing H₃32PO₄ for 30 min. (B). Human SMC were treated with 200 ng/ml of BMP-2 for the indicted times, and the uptake assays were carried out as described in A. Uptake values were normalized to cellular protein content. The results are presented as mean ± S.D. (n = 3). *Significant increase compared with vehicle (P < 0.05).

Fig. 4

BMP-2 upregulates Pit-1 mRNA and protein levels. (A) Human immortalized SMC were treated with 200 ng/ml of BMP-2 for 2 hrs, total RNA was isolated and Pit-1 mRNA levels were determined by quantitative real-time PCR. Pit-1 mRNA levels were normalized to 18S rRNA levels. Data are expressed as means ± SD (n=3). *P< 0.05. (B) Human immortalized SMC were treated with 200 ng/ml of BMP-2 for 12 hrs, protein lysates were prepared and Pit-1 and β-tubulin were detected by immunoblot analysis. (C) Pit-1 protein levels were quantified and normalized to β-tubulin levels. Data are presented as the percentage of SMC treated with vehicle.

Figure 5

Phosphonoformic acid blocks BMP-2 induced calcification. Human immortalized SMC were incubated with 200 ng/ml of human recombinant BMP-2 in the presence of 0.5 mM of PFA for 10 days. Calcium content of the extracellular matrix is given as mean ± S.D. (n = 3). *P < 0.05.

Fig. 6

BMP-2 induces osteogenic phenotype in human SMC. Human primary SMC were treated with 200 ng/ml of human recombinant BMP-2 for 2 days. Total RNA was isolated and human Runx2 (A) and SM22 (B) mRNA levels were quantitated using real-time PCR, and normalized to 18S rRNA levels. P< 0.05