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Am J Physiol Renal Physiol 293: F1592-F1604, 2007. First published August 22, 2007; doi:10.1152/ajprenal.00492.2006
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Elevated phosphorus modulates vitamin D receptor-mediated gene expression in human vascular smooth muscle cells

J. Ruth Wu-Wong, Masaki Nakane, Junli Ma, Xiaoan Ruan, and Paul E. Kroeger

Abbott Laboratories, Abbott Park, Illinois

Submitted 13 December 2006 ; accepted in final form 21 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clinical observations show that an increase in serum inorganic phosphorus (Pi) is linked to higher cardiovascular (CV) mortality, while vitamin D receptor (VDR) agonist therapy is associated with survival benefit in stage 5 chronic kidney disease. Smooth muscle cells (SMCs) play an important role in CV pathophysiology, but the interaction between Pi and the VDR signaling pathway in SMCs is not known. Real-time RT-PCR studies revealed that elevated Pi (2.06 mM) modulated VDR-mediated regulation of a panel of genes including thrombomodulin and osteopontin in SMCs. DNA microarray results demonstrated that increasing Pi from 0.9 to 2.06 mM exerted a widespread modulating effect on VDR-mediated gene expression. A total of 325 target genes were affected by paricalcitol at 0.9 mM Pi, with 195 up- and 130 downregulated. The number of target genes affected by paricalcitol at 2.06 mM Pi decreased to 86, with 55 up- and 31 downregulated. VDR-mediated gene expression in As4.1 cells (a juxtaglomerular cell-like cell line derived from kidney tumors in SV40 T-antigen transgenic mice) and peroxisome proliferator-activated receptor (PPAR){gamma}-mediated gene expression in SMCs were also altered by elevated Pi, suggesting that the observation is not unique to VDR in SMCs. Mechanism analysis showed that elevated Pi had no significant effect on VDR or PPAR{gamma} protein level but altered the cytosolic vs. nuclear distribution of NF-{kappa}B or nuclear receptor corepressor 1 (NCoR1). Our results demonstrate for the first time that elevated Pi affects VDR-mediated gene expression in human coronary artery SMCs and the effect is not limited to VDR in SMCs.

paricalcitol; hyperphosphatemia; microarray; gene expression

CLINICAL OBSERVATIONS SHOW that elevated serum inorganic phosphorus (Pi), which is often observed in chronic kidney disease (CKD), contributes to higher mortality in stage 5 CKD patients (24). It is generally believed that, besides being a risk factor for secondary hyperparathyroidism and renal osteodystrophy, hyperphosphatemia accelerates the vascular calcification process (6, 20, 24). Vascular calcification, the deposition of calcium and phosphate mineral in arteries, has emerged as an important risk factor for cardiovascular (CV) morbidity and mortality (9). Two distinct forms of calcification have been reported in the vessels (10, 26): intimal calcification is often associated with inflammation and atherosclerosis (10), whereas medial calcification occurs in smooth muscle cells (SMCs) (26). CKD patients, who experience a high mortality rate from CV diseases (8), have two- to fivefold more coronary artery medial calcification than age- and sex-matched individuals (3, 17, 25). During the past few years, there has been intense interest in understanding the effect of phosphorus on SMC calcification.

1,25-Dihydroxyvitamin D3 [1{alpha},25(OH)2D3, calcitriol] and its analogs such as 19-nor-1{alpha},25(OH)2D2 (paricalcitol) that activate vitamin D receptor (VDR) are commonly used to manage hyperparathyroidism secondary to CKD (16). Retrospective clinical observations show that VDR agonist or activator (VDRA) therapy is associated with a survival benefit for stage 5 CKD patients, in an effectiveness order of paricalcitol > calcitriol > no VDRA therapy, independent of parathyroid hormone (PTH), Pi, and calcium levels (18, 31, 32); the survival benefit of VDRA therapy is associated with a decrease in CV-related mortality (28). More recent studies using time-dependent analyses suggest that the survival benefit is in the order of intravenous paricalcitol > intravenous calcitriol > oral VDRA = no VDRA (12, 41). Although data from clinical studies demonstrate the potential benefit of VDRA therapy in mortality in CKD, the mechanism of action is largely unknown. Also, the observation that VDRA therapy is associated with survival benefit in CKD seems contradictory to the general perception that these drugs may elevate serum Ca, Pi, and calcium-phosphate product (CaxPi), resulting in medial layer calcification in vessels. Furthermore, very little is known about the interaction between elevated Pi and the VDR signaling pathway.

We showed previously (38) that Pi induces Ca accumulation in human coronary artery SMCs in a time- and dose-dependent manner, with a >25-fold increase in cellular Ca content after cells are cultured in medium containing 2.06 mM Pi for 5 days. However, VDR activators such as calcitriol and paricalcitol up to 100 nM have no significant effect on cellular Ca accumulation (38). We also showed (35) that calcitriol and paricalcitol are equally potent in modulating the expression of various genes such as thrombomodulin (TM), thrombospondin-1 (THBS1), and plasminogen activator inhibitor-1 (PAI-1) in human SMCs. TM is a monomeric transmembrane protein that serves as a cell surface receptor for thrombin (13), while THBS1 is a large glycoprotein that is released into the extracellular matrix by several cell types including SMCs (23) and PAI-1 is one of the risk markers for coronary heart disease (21). The three proteins have been shown to play roles in fibrinolysis and thrombogenicity.

In this study, we first compared the effect of paricalcitol at 2.06 vs. 0.9 mM Pi on the expression of a panel of genes including TM, THBS1, PAI, and some calcification-related genes. To investigate further, we employed DNA microarray technology to compare VDR-mediated gene expression profiles at 0.9 vs. 2.06 mM Pi in SMCs. To understand whether the impact of elevated Pi was unique to VDR in SMCs, we then examined the effect of 2.06 mM Pi on VDR-mediated gene expression in As4.1 cells, a juxtaglomerular (JG) cell-like cell line derived from kidney tumors in SV40 T-antigen transgenic mice. Furthermore, to check whether elevated Pi also affects the signaling pathway of other nuclear receptors in SMCs, we studied peroxisome proliferator-activated receptor (PPAR){gamma}-mediated effects because SMCs express PPAR{gamma}, Pex11a, and Cpt1a and it was shown previously that rosiglitazone, a PPAR{gamma} agonist, modulates the expression of Pex11a and Cpt1a (34). Our results clearly demonstrate that elevated Pi affects gene expression mediated by VDR in human coronary artery SMCs and the effect is not limited to VDR in SMCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Paricalcitol (Zemplar) was from Abbott Laboratories. Other reagents were of analytical grade.

Cell culture. Primary cultures of human coronary artery SMCs (Cambrex, East Rutherford, NJ or PromoCell, Heidelberg, Germany) were grown in SmGM-2 containing 5.5 mM glucose, 5% fetal bovine serum (FBS), 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, 5 µg/ml insulin, 2 ng/ml human (h)FGF, and 0.5 ng/ml hEGF at 37°C in a humidified 5% CO2-95% air atmosphere. Cells were cultured to >80% confluence and used within five passages. More than 10 different lots of SMCs from 2 different suppliers were used in this study, which might have caused data variability from experiment to experiment. As4.1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) with 10% FBS.

Real-time RT-PCR. Real-time RT-PCR was performed with the MyiQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Each sample had a final volume of 25 µl containing 100 ng of cDNA, each of the forward and reverse PCR primers at 0.4 mM, and 0.1 mM TaqMan probe for the gene of interest (Applied Biosystems). Temperature conditions consisted of a step of 5 min at 95°C, followed by 40 cycles of 60°C for 1 min and 95°C for 15 s. Data were collected during each extension phase of the PCR reaction and analyzed with the Bio-Rad software package. Threshold cycles were determined for each gene.

Microarray. Total RNA was extracted from human coronary artery SMCs that were treated with control or 100 nM paricalcitol in serum-free medium containing 0.9 or 2.06 mM phosphorus for 5 days. The RNAs were intact as judged by Agilent 2100 analysis. One microgram of total RNA from each sample was used to prepare biotin-labeled cRNA target with standard Affymetrix protocols. Prepared cRNA targets were of good quality and quantity. The Affymetrix human chip U133Av2 was used (22,000+ probe sets), and 10 µg of cRNA target was applied to each array. After hybridization and chip scanning, the quality control data report (i.e., scaling factor, GAPDH 5'-to-3' ratio, noise, background) demonstrated that every array passed all quality criteria. Scanned images were loaded into the Rosetta Resolver 4.0 database and processed with the Resolver Affymetrix error model. Within Resolver the sample replicates (n = 3 for each condition) were informatically combined and ratios were constructed relative to the combined vehicle control samples. A combination of hierarchical clustering, Gene Ontology analysis, and pathway mapping were used to assess the function of the regulated genes.

Preparation of cell extract. Cells were collected, and the cell volume was measured. The cytoplasmic and nuclear extracts were prepared with Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL). The samples were stored at –80°C until use.

SDS-PAGE and Western blot analysis. Cells (1 x 106 cells per sample) or cell extract preparations were solubilized in SDS-PAGE sample buffer (Invitrogen), and the protein content in each sample was determined by the Pierce bicinchoninic acid protein assay. Samples were resolved by SDS-PAGE using a 4–12% NuPAGE gel (Invitrogen), and proteins were electrophoretically transferred to polyvinylidene difluoride membrane for Western blotting. The membrane was blocked for 1 h at 25°C with 5% nonfat dry milk in PBS-Tween (PBS-T) and then incubated with a rabbit anti-osteoprotegerin (OPG) polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), a mouse anti-PAI-1 monoclonal antibody (1,000-fold dilution, Santa Cruz Biotechnology), a mouse anti-THBS1 monoclonal antibody (2,000-fold dilution, Calbiochem, La Jolla, CA), a mouse anti-TM monoclonal antibody (2,000-fold dilution, Santa Cruz Biotechnology), a mouse anti-VDR monoclonal antibody (1:500 dilution), a mouse anti-PPAR{gamma} (1:200 dilution) monoclonal antibody (Santa Cruz Biotechnology), a rabbit anti-NF-{kappa}B (p65 subunit) polyclonal antibody (1:200 dilution, Cell Signaling Technology, Danvers, MA), or a rabbit anti-nuclear receptor corepressor 1 (NCoR1) polyclonal antibody (1:200 dilution, Abcam, Cambridge, MA) in PBS-T overnight at 4°C. The membrane was washed with PBS-T and incubated with a horseradish peroxidase-labeled anti-mouse (for PAI-1, THBS1, TM, VDR, and PPAR{gamma}) or anti-rabbit (for OPG, NF-{kappa}B, and NCoR1) second antibody for 1 h at 25°C. The membrane was then incubated with detection reagent (SuperSignal WestPico, Pierce). Specific bands were visualized by exposing the paper to Kodak BioMax films. Band intensity was quantified by Quantity One (Bio-Rad).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We first examined the effect of paricalcitol on the expression of a panel of genes at 0.9 vs. 2.06 mM Pi in SMCs. Fig. 1A shows that paricalcitol at 100 nM induced the expression of TM by 328% in cells cultured in normal medium containing 0.9 mM Pi, but its effect was significantly reduced when the Pi concentration was increased to 2.06 mM. Pi alone (at 2.06 mM) had no effect on the basal expression of TM. Figure 1B shows that paricalcitol at 100 nM suppressed the expression of THBS1 at 0.9 mM Pi by 50%. When the Pi concentration was increased to 2.06 mM, the expression of THBS1 was reduced. However, the suppressive effect of paricalcitol on THBS1 expression was still observed. Figure 1C shows that paricalcitol at 100 nM suppressed the expression of PAI-1 at 0.9 mM Pi by 29%, and its effect was not affected by 2.06 mM Pi. We have also tested calcitriol, and the observations were very similar (data not shown). The effects of VDRA on these three genes at 0.9 mM Pi are consistent with those reported previously (37).


Figure 1
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  		Fig. 1. Effect of paricalcitol on the expression of a panel of genes in human coronary artery smooth muscle cells (SMCs) at 0.9 vs. 2.06 mM inorganic phosphorus (Pi). A: thrombomodulin (TM). B: thrombospondin-1 (THBS1). C: plasminogen activator inhibitor-1 (PAI-1). D: matrix Gla protein (MGP). E: osteoprotegerin (OPG). F: osteopontin. G: core binding factor {alpha}1 (Cbfa1). H: collagen type I {alpha}1 (COL1A1). I: alkaline phosphatase (ALP). SMCs were cultured in medium containing 0.9 or 2.06 mM Pi in the presence or absence of 100 nM paricalcitol for 6 days. Medium containing the appropriate test agents was changed every 3 days. RNA was isolated with TRIzol (Invitrogen) followed by a Qiagen RNeasy minikit. Real-time RT-PCR was performed as described in MATERIALS AND METHODS. The mRNA expression level of each test gene was first normalized to the GAPDH mRNA level and then calculated as % of control (0.9 mM Pi without paricalcitol = 100%). Values shown are means ± SD (n = 4). Unpaired t-test with 95% confidence intervals of difference was performed for statistical comparisons. **P < 0.01 vs. control (no drug treatment) at corresponding Pi concentrations; ++P < 0.01, 0.9 vs. 2.06 mM Pi-control; ##P < 0.01, 0.9 vs. 2.06 mM Pi-paricalcitol treated. Results shown are representative of a minimum of 3 independent experiments.

 
We also examined six genes that have been implicated in the vascular calcification process. As shown in Fig. 1, D–I, when Pi was increased from 0.9 to 2.06 mM, only the OPG level was modestly increased, which resulted in a seemingly augmented paricalcitol-mediated suppressing effect. Elevated Pi had no significant effect on the basal level of matrix Gla protein (MGP), osteopontin, core binding factor {alpha}1 (Cbfa1), collagen type I {alpha}1 (COL1A1), and alkaline phosphatase (ALP). On the other hand, at 0.9 mM Pi, paricalcitol upregulated MGP, osteopontin, COL1A1, and ALP, had no significant effect on Cbfa1, and suppressed the expression of OPG. Except for osteopontin, the effects of paricalcitol on these genes at 0.9 mM Pi were modest. At 2.06 mM Pi, paricalcitol-mediated upregulation of osteopontin was reduced, while its effect on modulating MGP, COL1A1, and ALP seemed unaltered.

These results suggest that the combined effects of elevated Pi and paricalcitol on gene expression could be categorized as follows: 1) elevated Pi had no effect and also did not impact paricalcitol-mediated effect (e.g., PAI-1, MGP, COL1A1, and ALP); 2) elevated Pi by itself had no effect on gene expression, but paricalcitol-mediated effects were reduced at elevated Pi, such as in the case of TM and osteopontin; 3) elevated Pi upregulated the gene, which resulted in a seemingly augmented paricalcitol-mediated effect (e.g., OPG); and 4) elevated Pi downregulated the gene, but did not appear to impact paricalcitol-mediated effects (e.g., THBS1).

To study the combined effects of Pi and paricalcitol in detail, we selected one gene from each of the four categories for further analysis. Figure 2A shows that, at 0.9 mM Pi, paricalcitol modulated the expression of MGP in a dose-dependent manner with an EC50 of 10 nM; its effect was not significantly different at 2.06 mM Pi (EC50 = 25 nM). Figure 2B shows that paricalcitol upregulated TM in a dose-dependent manner at both 0.9 and 2.06 mM Pi, with EC50 of 98 and 24 nM, respectively. Elevated Pi dampened the upregulation of TM induced by paricalcitol at 100 and 1,000 nM but not at the two lower concentrations (1 and 10 nM). Figure 2C shows that the OPG level was higher at 2.06 mM Pi. Paricalcitol suppressed OPG at both 0.9 and 2.06 mM Pi, with EC50 of 16 and 11 nM, respectively. Paricalcitol at 100 nM suppressed OPG expression by 65% at 0.9 mM Pi and by 73% at 2.06 mM Pi. Figure 2D shows that the THBS1 level was lower at 2.06 mM Pi. Paricalcitol suppressed THBS1 at both 0.9 and 2.06 mM Pi, with EC50 of 11 and 38 nM, respectively. Paricalcitol at 100 nM suppressed THBS1 expression by 91% and 79% at 0.9 and 2.06 mM Pi, respectively. These results suggest that the modulating effect of elevated Pi on VDR-mediated gene expression appears to be dose- and gene dependent.


Figure 2
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  		Fig. 2. Dose-dependent effect of paricalcitol on the expression of selected genes in human coronary artery SMCs at 0.9 vs. 2.06 mM Pi. A: MGP. B: TM. C: OPG. D: THBS1. SMCs were treated and real-time RT-PCR performed as in Fig. 1. The mRNA expression level of each test gene was first normalized to the GAPDH mRNA level and then calculated as % of control (0.9 mM Pi without paricalcitol = 100%). Values shown are means ± SD (n = 4). One-way ANOVA Dunnett test with 95% confidence intervals of difference was performed for statistical comparisons, At 0.9 mM Pi: #P < 0.05, ##P < 0.01 vs. control (no paricalcitol). At 2.06 mM Pi: +P < 0.05, ++P < 0.01 vs. control (no paricalcitol). To check the impact of elevated Pi on paricalcitol-mediated effects, unpaired t-test with 95% confidence intervals of difference was performed for statistical comparisons for 0.9 vs. 2.06 mM Pi at a corresponding paricalcitol concentration: *P < 0.05, **P < 0.01, ***P < 0.001. Results shown are representative of 3–6 (MGP: 4, TM: 3, OPG: 6, THBS1: 3) independent experiments.

 
To further extend the studies, we determined the protein levels of selected genes, namely, OPG, PAI-1, TM, and THBS1. Figure 3 shows that increasing Pi from 0.9 to 2.06 mM caused a reduction in the protein levels of OPG (both the monomer and the homodimer at 60 and 120 kDa, respectively) and THBS1 but had no significant effect on PAI-1. The TM level in the absence of paricalcitol was too low for detection. Paricalcitol at 100 nM significantly suppressed OPG, PAI-1, and THBS1 at both 0.9 and 2.06 mM Pi. Consistent with the real-time RT-PCR results, paricalcitol at 100 nM greatly induced the expression of TM protein at 0.9 mM Pi, but the paricalcitol-mediated increase in the TM protein level was reduced at 2.06 mM Pi.


Figure 3
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  		Fig. 3. Effect of paricalcitol on protein levels of selected genes at 0.9 vs. 2.06 mM Pi. SMCs were treated as in Fig. 1. Cells were then solubilized and Western blotting performed as described in MATERIALS AND METHODS. The specific bands were visualized, and the density of each band was measured. The band was then normalized to the protein content in each sample. Similar results were obtained when the density of each band was measured and normalized to the actin level in each sample. Results shown are representative of 2 independent experiments. C, control, no drug treatment.

 
To assess the breadth of the impact of elevated Pi on VDR-mediated gene expression, we treated SMCs with 100 nM paricalcitol at 0.9 or 2.06 mM Pi and then subjected the samples to DNA microarray analysis. When 2.06 and 0.9 mM Pi-treated samples (in the absence of paricalcitol) were compared directly, only six genes were identified based on a twofold change in average difference as cutoff with P < 0.01 for significantly modulated expression (Table 1). When the criteria were relaxed to a 1.5-fold change as cutoff with P < 0.01, then 18 sequences were identified (Table 1).


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  		Table 1. Genes differentially regulated by 2.06 mM Pi (vs. 0.9 mM Pi) in the absence of paricalcitol based on a 1.5-fold change and P < 0.01

 
As a comparison, in cells treated with paricalcitol in normal medium containing 0.9 mM Pi, a total of 325 target sequences were identified, with 195 and 130 genes up- and downregulated, respectively (Fig. 4A). When the Pi concentration was increased to 2.06 mM, the number of target sequences affected by paricalcitol decreased to 86, with 55 up- and 31 downregulated. Figure 4B shows the hierarchical clustering of sequences regulated by paricalcitol at 0.9 mM vs. 2.06 mM Pi. It is evident that there is a significant difference between these two sets of data. Indeed, only 31 paricalcitol-modulated sequences were shared under 0.9 and 2.06 mM Pi conditions (Fig. 4A). Table 2 shows the 31 paricalcitol-mediated sequences that were shared at 0.9 and 2.06 mM Pi based on a twofold change with P < 0.01. Of these 31 sequences, only 8 sequences (Table 2) were regulated by paricalcitol to the same degree at 0.9 and 2.06 mM Pi (<1.5-fold difference). For the remaining 23 sequences (Table 2), a change of >1.5-fold was evident when comparing the paricalcitol effect at 0.9 vs. 2.06 mM Pi. The microarray data from the paricalcitol-treated groups were then analyzed, using DAVID 2.1 to assess the general effects of paricalcitol on intracellular signaling and metabolic pathways. Gene Ontology categories with at least three genes and P < 0.001 regulated by paricalcitol at 0.9 or 2.06 mM Pi are shown in Table 3. For the 2.06 mM Pi + paricalcitol sample set, only seven clusters were identified: antioxidant activity, extracellular matrix structural constituent, homeostasis, icosanoid biosynthesis, icosanoid metabolism, organogenesis, and receptor binding (Table 3). In the 0.9 mM Pi + paricalcitol sample set, a number of functionally related clusters were revealed (Table 3). These results demonstrate that 2.06 mM Pi exhibits a broad modulating effect on paricalcitol-regulated gene expression.


Figure 4
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  		Fig. 4. Effects of paricalcitol on gene expression at 0.9 vs. 2.06 mM Pi from the microarray study. The genes regulated by 100 nM paricalcitol at 0.9 or 2.06 mM Pi (A) are shown in hierarchical clustering using a 2-fold change in average difference as cutoff with P < 0.01 (B).

 

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  		Table 2. Paricalcitol-mediated sequences shared at 0.9 vs. 2.06 mM Pi

 

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  		Table 3. Pathways modulated by paricalcitol at 2.06 mM vs. 0.9 mM Pi

 
Selected paricalcitol-modulated genes that are known to be involved in CV pathophysiology are shown in Table 4. The top portion of the table shows the sequences modulated by paricalcitol at 0.9 mM Pi but not at 2.06 mM Pi. Those sequences affected by paricalcitol at 2.06 mM Pi alone are shown in the middle portion, while the sequences modulated by paricalcitol at both 0.9 and 2.06 mM Pi are listed in the bottom portion. The results demonstrate that, in general, elevated Pi significantly impacted paricalcitol-modulated gene expression. Interestingly, a subset of genes were responsive to paricalcitol treatment only at 2.06 mM Pi.


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  		Table 4. Effects of paricalcitol on selected cardiovascular-related sequences at 0.9 vs. 2.06 mM Pi

 
To investigate whether the experimental conditions used in the aforementioned studies might fail to catch the effect of elevated Pi on gene expression, SMCs were cultured in medium containing either 0.9 or 2.06 mM Pi for different periods of time, and selected genes were examined by real-time RT-PCR. Figure 5 shows that 2.06 mM Pi had no significant effect on MGP and ALP. However, elevated Pi significantly increased the OPG level as early as day 1, and the effect continued throughout the study period. Elevated Pi seemed to modestly increase COL1A1, but the effect was not consistent at the different time points.


Figure 5
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  		Fig. 5. Time-course analysis of selected genes in human coronary artery SMCs at 0.9 vs. 2.06 mM Pi. SMCs were cultured in medium containing 0.9 or 2.06 mM Pi for different periods of time. Medium containing the appropriate test agents was changed every 3 days. Real-time RT-PCR was performed as in Fig. 1. The mRNA expression level of each test gene was first normalized to the GAPDH mRNA level, and then the level at 2.06 mM Pi was calculated as % of control (0.9 mM Pi at the corresponding time point = 100%). Unpaired t-test with 95% confidence intervals of difference was performed for statistical comparisons. *P < 0.05, **P < 0.01 vs. 0.9 mM Pi at the corresponding time point.

 
To explore whether this observation is unique for SMCs, we examined VDR-modulated gene expression in As4.1 cells, a JG cell-like cell line derived from kidney tumors in SV40 T-antigen transgenic mice with a high level of renin synthesis. Previously, it was shown that calcitriol and paricalcitol modulate the expression of a variety of genes including renin in As4.1 cells (14, 19), and both drugs were equipotent in suppressing renin expression. In this study, we randomly selected three genes for examination. Figure 6 shows that paricalcitol at 10 and 100 nM downregulated renin but upregulated CYP24A1 and IL-1{alpha} at 0.9 mM Pi. When Pi was increased to 2.06 mM the expression level of renin was significantly increased, while the IL-1{alpha} level was decreased. The effect of paricalcitol on CYP24A1 and IL-1{alpha} was greatly modulated by 2.06 mM Pi, but its effect on suppressing renin was still evident. Furthermore, the paricalcitol effect on inhibition of renin was higher at 2.06 mM Pi (~70% suppression at 100 nM) than at 0.9 mM Pi (~50%). It is perhaps of interest to note that the effect of paricalcitol on inducing CYP24A1 was significantly impacted by elevated Pi in both As4.1 cells and SMCs (Fig. 6 and Table 2). The results from As4.1 cells suggest that the effect of elevated Pi on the expression of VDR-mediated gene expression is not unique to SMCs.


Figure 6
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  		Fig. 6. Effect of paricalcitol on expression of selected genes in As4.1 cells at 0.9 vs. 2.06 mM Pi. As4.1 cells were cultured in DMEM with 10% fetal bovine serum containing 0.9 or 2.06 mM Pi for 4 days. Cells were then transfected with the pcDNA-hVDR plasmid (kindly provided by Dr. Yan Chun Li, University of Chicago) with Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were treated with 10 or 100 nM paricalcitol for another 24 h. Samples were processed and real-time RT-PCR performed as in Fig. 1. The mRNA expression level was first normalized to the GAPDH mRNA level and then calculated as % of control (0.9 mM Pi without paricalcitol = 100%). Values shown are means ± SD (n = 4). One-way ANOVA Dunnett test with 95% confidence intervals of difference was performed for statistical comparisons. **P < 0.01 vs. control (no drug treatment) at corresponding Pi concentrations; ##P < 0.01: 0.9 vs. 2.06 mM Pi control.

 
To investigate whether the effect of Pi is unique for VDR, we then examined genes in SMCs that are regulated by PPAR{gamma}, another nuclear receptor. It was previously shown that rosiglitazone, a PPAR{gamma} agonist, modulates the expression of a variety of genes in macrophages (34). We examined the expression of several of those genes in SMCs and found that SMCs expressed Pex11a and Cpt1a. When SMCs were treated with 30 µM rosiglitazone at 0.9 or 2.06 mM Pi for 6 days, rosiglitazone had a modest effect in upregulating the two genes at 0.9 mM Pi (Fig. 7). However, at 2.06 mM Pi, rosiglitazone significantly suppressed the expression of Pex11a and Cpt1a (90% and 73%, respectively). These results suggest that elevated Pi also affected the gene expression pattern mediated by PPAR{gamma}.


Figure 7
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  		Fig. 7. Effect of rosiglitazone on expression of PEX11A and CPT1A in human smooth muscle cells at 0.9 vs. 2.06 mM Pi. SMCs were cultured in medium containing 0.9 or 2.06 mM Pi in the presence or absence of 30 µM rosiglitazone for 6 days. Samples were processed and real-time RT-PCR performed as in Fig. 1. The mRNA expression level was first normalized to the GAPDH mRNA level and then calculated as % of control (0.9 mM Pi without rosiglitazone = 100%). Values shown are means ± SD (n = 4). Unpaired t-test with 95% confidence intervals of difference was performed for statistical comparisons. *P < 0.05, ***P < 0.001 vs. control (no drug treatment) at corresponding Pi concentrations.

 
To further study the mechanism of how elevated Pi affects gene expression mediated by VDR, we examined the VDR protein level in SMCs cultured at 0.9 and 2.06 mM Pi. Results from Western blotting analysis demonstrated that the VDR level was not significantly altered at 2.06 mM Pi (Fig. 8A). Figure 8B shows that paricalcitol treatment resulted in an increase in the VDR protein level at both 0.9 and 2.06 mM Pi, likely because of the stabilization of VDR (36). We next checked whether elevated Pi affects agonist-mediated translocation of VDR. Figure 8C shows that after cells were incubated with paricalcitol at 0.9 or 2.06 mM Pi for 5 days, VDR was present in the nuclear extracts of paricalcitol-treated cells, but no significant difference was observed at 0.9 vs. 2.06 mM Pi. Since paricalcitol treatment may affect VDR stability and translocation over the 5-day incubation period, we examined VDR translocation after 24 h of paricalcitol treatment. Cells were treated with 0.9 or 2.06 mM Pi for 5 days, followed by treatment with 100 nM paricalcitol for 24 h. Figure 8C shows that VDR was detected in the nuclear extracts of paricalcitol-treated cells but was not detectable in the control (untreated) samples. However, no significant difference was observed between 0.9 and 2.06 mM Pi.


Figure 8
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  		Fig. 8. Effect of elevated Pi and paricalcitol on vitamin D receptor (VDR) protein levels. A: SMCs were cultured in medium containing 0.9 or 2.06 mM Pi for 3 or 5 days. B: SMCs were cultured in medium containing 0.9 or 2.06 mM Pi for 6 days in the presence of different concentrations of paricalcitol. Cells were solubilized and Western blotting performed as described in MATERIALS AND METHODS. C: SMCs were cultured in medium containing 0.9 or 2.06 mM Pi in the presence or absence of 100 nM paricalcitol for 5 days. Alternatively, SMCs were cultured in medium containing 0.9 or 2.06 mM Pi for 5 days and then treated with or without 100 nM paricalcitol for 1 day. Nuclear extracts were prepared and Western blotting performed as described in MATERIALS AND METHODS. The specific bands were visualized, and the density of each band was measured and normalized to the protein level in each sample.

 
We also examined the PPAR{gamma} protein level in SMCs cultured at 0.9 and 2.06 mM Pi. Results from Western blotting analysis demonstrated that PPAR{gamma} was detected in SMCs and the total cellular protein level was not significantly different after 3 or 5 days at 0.9 vs. 2.06 mM Pi (data not shown). PPAR{gamma} was detected in the nuclear extracts but was not detectable in the cytosolic fractions. However, no significant difference in the nuclear PPAR{gamma} protein level was observed between 0.9 and 2.06 mM Pi (data not shown).

It is possible that elevated Pi affected the function of other transcription factors that are components of the VDR or PPAR{gamma} transcriptional complex. We chose the p65 subunit of NF-{kappa}B and NCoR1 for additional studies based on the fact that both have been linked to the VDR and/or PPAR{gamma} signaling pathway (1, 15, 33). Previously, it was shown that VDRAs affected the I{kappa}B-NF-{kappa}B interaction within the first 24 h of incubation in macrophages (5) and in pancreatic islets (7). Figure 9A shows that the total level of NF-{kappa}B in SMCs was not significantly different among the four samples. Figure 9B shows that NF-{kappa}B was detected mainly in the cytosol at 0.9 mM Pi. When the Pi level increased from 0.9 to 2.06 mM, the level of NF-{kappa}B in the nuclear extract increased by 13-fold, while the cytosolic NF-{kappa}B level decreased by ~40%. No significant difference was observed between samples with or without paricalcitol at 100 nM. Figure 9C shows that the total level of NCoR1 in SMCs was slightly higher in the 0.9 mM Pi control sample (no paricalcitol treatment). Paricalcitol treatment appeared to decrease the NCoR1 level at both 0.9 and 2.06 mM Pi. Figure 9D shows that NCoR1 was detected mainly in the nuclear extract at 0.9 mM Pi. When the Pi level increased from 0.9 to 2.06 mM, the nuclear NCoR1 level increased by threefold while the cytosolic level decreased by ~90%. Paricalcitol treatment appeared to decrease the cytosolic NCoR1 level at 0.9 mM Pi and increase the nuclear NCoR1 level at 2.06 mM Pi.


Figure 9
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  		Fig. 9. Effect of elevated Pi and paricalcitol on the cytosolic vs. nuclear level of NF-{kappa}B (A and B) or nuclear receptor corepressor 1 (NCoR1) (C and D). A and C: SMCs were treated with or without 100 nM paricalcitol in medium containing 0.9 or 2.06 mM Pi for 6 days. B and D: SMCs were cultured in medium containing 0.9 or 2.06 mM Pi for 5 days and then treated with or without 100 nM paricalcitol for 24 h. Cells were solubilized. Nuclear and cytosolic extracts were prepared. Western blotting was performed as described in MATERIALS AND METHODS. The protein level in each sample was determined to ensure that same amounts of protein were used in SDS-PAGE. The specific bands were visualized, and the density of each band was measured and normalized to the protein level in each sample.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, our data clearly demonstrate that increasing Pi from 0.9 to 2.06 mM affected VDR-mediated gene expression in SMCs. In addition, the PPAR{gamma} and As4.1 data suggest that the effect of elevated Pi is not limited to VDR in SMCs.

Because of our previous finding (38) that Pi at 2.06 mM for 5 days induces a >25-fold increase in cellular Ca content in human coronary artery SMCs, it was surprising to us that, using modest statistical cutoff settings in the microarray study (either a 1.5-fold or 2-fold change in average difference with P < 0.01), we observed a limited set of genes affected by elevated Pi. The microarray observations are consistent with the results from real-time RT-PCR studies examining a panel of selected genes such as ALP, Cbfa1, PAI-1, osteopontin, TM, etc. with different lots of SMCs over a period of 2 yr. A search in the literature regarding the effect of Pi on gene expression yielded rather limited information. In one study using SMCs isolated from human fetal or newborn aortas, cells treated with 2.6 mM Pi for 10 days exhibited a modest increase in Cbfa1 and ALP (<1.5-fold; Ref. 40). However, Steitz et al. (30) reported that ALP and osteocalcin expression in bovine aortic SMCs were significantly induced by 5 mM Pi after 10 days of treatment, but their levels were not much different from control after 4 days of treatment. In that same study, elevated Pi, in the form of beta-glycerophosphate, had a profound effect on stimulating Cbfa1 expression (>10-fold after 10 days) but no significant effect after 1 day of treatment. Jono et al. (11) reported that in human fetal SMCs calcification medium containing 2 mM Pi was able to induce a twofold increase in both the osteocalcin and Cbfa1 genes after 24 h of treatment. It is likely that the effect of elevated Pi on regulating gene expression in SMCs is dependent on cell type and/or experimental conditions. It is also possible that our experimental conditions (2.06 mM Pi for 5 days), although adequate in inducing Ca accumulation in SMCs and in modulating VDR-mediated gene expression, were not optimal for detecting the direct effect of elevated Pi on gene expression.

We previously reported (39) the effects of paricalcitol and calcitriol on gene expression profiling in human coronary artery SMCs under proliferating or resting conditions at 0.9 mM Pi. In both studies, SMCs were treated with a high concentration of paricalcitol (100 nM) for 30 h to reveal a majority of the genes that are likely regulated by VDR. While the studies do not differentiate genes that are directly regulated vs. those that are indirectly regulated by VDR, it allows us to identify many genes that are known targets of vitamin D analogs plus a large number of additional genes that were not previously known to be affected by vitamin D analogs. The same rationale was employed here, where cells were treated with 100 nM paricalcitol at 0.9 vs. 2.06 mM Pi in the microarray analysis. When we compared the present study with the previous ones, minimal differences (<18%) were observed for the effect of paricalcitol on gene expression in cells cultured in normal medium. For example, we reported previously that paricalcitol upregulated angiopoietin 1 (ANGPT1), endothelin receptor type B (EDNRB), thrombomodulin (THBD or TM), transforming growth factor-beta-3 (TGFB3), etc.; similar changes were observed in this study. Also, we showed previously that paricalcitol downregulated THBS1, OPG (TNFRSF11B), prostaglandin E synthase (PTGES), PAI-1 (SERPINE1), etc.; the same pattern was found in the present study.

The effects of paricalcitol on the CV-related sequences were greatly reduced when Pi was increased from 0.9 to 2.06 mM (Table 4). For example, many factors detrimental to the CV system, such as PAI-1, IL-6, IL-1beta, and IL-8, that were suppressed by paricalcitol at 0.9 mM Pi were no longer significantly modulated by paricalcitol at 2.06 mM Pi. This observation is consistent with the idea that elevated Pi has adverse effects, and it also offers a new mechanism besides vascular calcification to explain the effect of hyperphosphatemia on increasing the mortality risk in stage 5 CKD patients. Interestingly, a subset of genes are responsive to paricalcitol treatment only at high Pi, some of which are known to play roles in CV pathophysiology. For instance, NR4A1 and NR4A2 have been associated with protective effects in SMCs in models of vascular disease (2). IL-11, a growth factor that stimulates the growth of primitive megakaryocytic progenitor cells and the activity of mature megakaryocytes, is in use for the treatment of thrombocytopenia (27). An increase in these factors may be beneficial. PTGS2 (COX-2) is involved in the inflammation process, and suppression of PTGS2 is supposed to have positive effects for the CV system. However, recent clinical data have shown that patients taking COX-2 antagonists experienced a higher risk for an increased incidence of CV events (22). On the other hand, it is well known that targeted disruption of the APOE gene results in accelerated atherosclerosis (4), so the impact of VDR-mediated modulation of APOE (a 2-fold decrease) at elevated Pi is less certain. Together, our microarray results confirm that many genes linked to CV pathophysiology are modulated by VDR, while hyperphosphatemia exerts a broad modulating effect on VDR-mediated gene expression.

Regarding the mechanism of how elevated Pi affects VDR- or PPAR{gamma}-mediated gene expression, our results demonstrate that 2.06 mM Pi does not affect the level of VDR or PPAR{gamma} protein, and also does not have a significant effect on agonist-induced VDR translocation into the nucleus. These results are consistent with the observation that a subset of VDR-regulated genes were not affected by 2.06 mM Pi, suggesting that VDR function is at least partially intact. The observation on the cytoplasmic vs. nuclear distribution of NF-{kappa}B and NCoR1 suggests that elevated Pi impacts other transcription factors that have been shown to be present in the VDR or PPAR{gamma} transcriptional complex. It is known that for nuclear receptors to exert their effect on transcriptional programs they need to reside in a large complex containing various coactivators and corepressors. A subtle change in any of the cofactors could potentially lead to a change in function. For example, it was recently shown that an increase in NCoR1 correlates with suppressed regulation of VDR target gene (1). Also, Lu et al. (15) showed that, once the p65 subunit of NF-{kappa}B anchors itself to proteins within the VDR transcription complex, p65 disrupts VDR binding to SRC-1, thus altering the efficiency of VDR-mediated gene transcription. Elevated Pi, by increasing the nuclear NF-{kappa}B and NCoR1 levels, may impair transactivation of a subset of genes mediated by the VDR-SRC-1 pathway. However, this cannot fully explain the reduction in VDR-mediated effects observed for so many genes. Furthermore, the effect of elevated Pi on OPG protein was opposite to that on OPG mRNA (Figs. 2 and 3), suggesting that posttranscriptional regulation such as mRNA stability may be involved. It has been shown that elevated Pi affects PTH mRNA stability (29). Thus the cross talk between the pathways mediated by elevated Pi and VDR activation may be quite complex. Although it will require further analysis to elucidate the details of the mechanism, our data suggest that elevated Pi may have potential modulating effects on mRNA stability and also alter the function of some transcription factors and/or their cofactors, resulting in impaired assembly of the transcription complex mediated by VDR or PPAR{gamma}.

The observation made in this study that elevated Pi modulates gene expression mediated by VDR and PPAR{gamma} provides a new mechanism to explain the detrimental effect of hyperphosphatemia. On the other hand, results from both the SMC and As4.1 cell studies suggest that, even in the presence of elevated Pi, VDR can still function in regulating various genes. Moreover, in addition to genes expressed in SMCs, other genes outside of SMCs that are modulated by vitamin D analogs may also play a role in VDR-mediated benefits in kidney disease. Taking renin as an example, elevated Pi upregulated renin, but paricalcitol still showed a suppressive effect on renin expression at high Pi. Although it is beyond the scope of this report, it will be important to explore whether elevated Pi affects functional responses mediated by other nuclear receptors and/or G protein-coupled receptors. Also, the question of whether what we have observed in cultured cells in this study can be replicated in animal disease models remains unanswered. Future studies to further investigate the interaction between elevated Pi and VDR activation may lead to an understanding of how vitamin D analogs provide survival benefits even at elevated Pi with paricalcitol therapy associated with better survival in stage 5 CKD.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Wu-Wong, Department of Pharmacy Practice, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612 (e-mail: jrwuwong{at}uic.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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