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Am J Physiol Renal Physiol 293: F946-F955, 2007. First published May 30, 2007; doi:10.1152/ajprenal.00474.2006
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Extracellular calcium-sensing receptor is functionally expressed in human artery

Guerman Molostvov,1 Sean James,3 Simon Fletcher,4 Jeanette Bennett,2 Hendrik Lehnert,1 Rosemary Bland,2 and Daniel Zehnder1

1Clinical Sciences Research Institute and 2BioMedical Research Institute, University of Warwick, and Departments of 3Pathology and 4Nephrology, University Hospital Coventry and Warwickshire, Coventry, United Kingdom

Submitted 30 November 2006 ; accepted in final form 29 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Accelerated medial calcification is a major cause of premature cardiovascular mortality in patients with chronic kidney disease (CKD). Evidence suggests that extracellular concentration of Ca2+ and vascular smooth muscle cells may play a pivotal role in the pathogenesis of vascular calcification. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that is expressed in a range of tissues, but characterization of its expression and function in the cardiovascular system is limited. Here we report the expression of CaSR mRNA (RT-PCR) and protein (Western blotting and immunocytochemistry) in human aortic smooth muscle cells (HAoSMC). Treatment of HAoSMC with Ca2+ (0–5 mM; 0–30 min) or the CaSR agonists gentamycin and neomycin (0–300 µM; 0–30 min) resulted in a dose- and time-dependent phosphorylation of ERK1/2. Gentamycin- and neomycin-mediated ERK1/2 stimulation was inhibited by pretreatment with PD-98059, an ERK-activating kinase 1 (MEK1) inhibitor, confirming specificity of the observed effects. ERK1/2 activation was inhibited in HAoSMC, with CaSR expression knocked down by transfection with specific small-interference RNA, which confirmed that the observed neomycin/gentamycin-induced MEK1/ERK1/2 activation was mediated via the CaSR. CaSR mRNA and protein were also expressed in large and small arteries from normal subjects (kidney donors) and patients with end-stage renal disease (ESRD). The CaSR was detected in smooth muscle and endothelial cells. Expression was significantly lower in arteries from ESRD patients. In conclusion, these data not only demonstrate the presence of a functional CaSR in human artery but show a correlation between CaSR expression and progression of CKD.

vascular smooth muscle cell; calcification; chronic kidney disease

THE IMPORTANCE OF INTRACELLULAR Ca2+ in regulating multiple vascular smooth muscle cell (SMC) functions including vascular tone and blood flow is well characterized. However, the physiological role of extracellular Ca2+ and the Ca-sensing receptor (CaSR) in human vascular SMC is not well understood. The extracellular CaSR is a 1,078-amino acid cell surface protein, which was initially cloned and characterized in bovine parathyroid cells. It responds to changes in serum Ca2+ concentration by regulating the synthesis of parathyroid hormone (4). It belongs to the family C of the superfamily of seven-transmembrane receptors, also known as G protein-coupled receptors (5). Like other family members, it contains three structural domains: the extracellular domain, crucial for the interactions with extracellular Ca2+, the transmembrane domain containing seven hydrophobic helices that anchor it in the plasma membrane, and the cytosolic tail with regulatory protein kinase phosphorylation sites (12).

After identification of the CaSR in the parathyroid cells, a number of studies followed, which established the expression of this receptor in a broad range of cells and tissues not directly involved in mineral homeostasis, such as hematopoietic and immune cells, brain, gastrointestinal tract, and placenta (5). Binding of extracellular Ca2+ or other CaSR agonists and activation of the receptor trigger multiple intracellular signaling events, such as activation of phospholipase C, leading to the generation of second messengers diacylglycerol and inositol triphosphate, inhibition of adenylate cyclase, which suppresses intracellular concentration of cAMP, and activation of the MEK/ERK1/2 pathway (27). Utilizing these and other pathways, the CaSR can regulate such diverse processes as hormone secretion, gene expression, ion channel activity, proliferation, differentiation, apoptosis, and modulation of inflammation (6, 19, 27).

It is necessary to note that the current literature on CaSR expression and function in the human cardiovascular system is rather limited. CaSR mRNA and protein were recently identified in rat ventricular cardiomyocytes (28, 31). Importantly, the CaSR was functional, as it responded to stimulation with extracellular Ca2+, gadolinium, and spermine. Weston et al. (33) provided evidence in favor of CaSR expression in endothelial cells from rat mesenteric and porcine coronary arteries. Using allosteric modulators of the CaSR they also showed hyperpolarization of vascular SMC. A recently published report demonstrated the expression of a functional CaSR in human aortic endothelial cells (37).

Importantly, accumulating evidence suggests that the CaSR is functionally expressed in vascular SMC. Wonneberger et al. (34) showed the presence of CaSR mRNA in the gerbil spiral modiolar artery. Using Ca2+ and CaSR agonists, they induced a biphasic increase in intracellular Ca2+, which was likely to be localized in vascular SMC, since it was paralleled by a biphasic vasoconstriction. A similar observation was made in rat subcutaneous arteries (18). Finally, Smajilovic et al. (26) reported the expression of CaSR mRNA and protein in rat aortic vascular SMC and showed that extracellular Ca2+ stimulated proliferation of the cells, likely through the MEK1/ERK1/2 pathway.

Vascular SMC are thought to play an important role in the pathogenesis of vascular calcification. Accelerated medial arterial calcification is a serious problem in the treatment of patients with CKD and a major cause of premature cardiovascular death (10, 15). A number of studies suggest that vascular calcification is an active cell-mediated process similar to bone formation (3, 23). They demonstrated that vascular SMC cultured in vitro could convert to an osteo/chondrocytic phenotype and form calcified nodules in a manner similar to osteoblasts (3, 25, 30).

Important risk factors associated with cardiovascular disease and vascular calcification in CKD patients and shown to increase SMC calcification in patients (2) and in vitro (35) include elevated extracellular Ca2+, potentially by the activation of the CaSR expressed on vascular SMC.

Taken together, these findings indicate that the role of extracellular Ca2+ in SMC function is likely to be of great importance in the pathogenesis of vascular calcification. Therefore, our project was aimed to investigate expression and distribution of the CaSR in human vascular SMC and to examine its functional significance in patients with CKD. Here we demonstrate that the CaSR is expressed in human vascular SMC and is capable of mediating CaSR agonist-induced activation of the MEK1/ERK1/2 signaling pathway. Importantly, we have shown that CaSR expression tends to decline in arterial SMC of ESRD patients.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and tissue samples. HAoSMC were purchased from PromoCell and maintained in SMC growth medium 2 containing 5% FCS, 0.5 ng/ml epidermal growth factor, 2.0 ng/ml basic fibroblast growth factor, and 5 µg/ml insulin (PromoCell). The cells were grown under 5% CO2 at 37°C in medium renewed every 3 days. Confluent cells were detached by trypsin/EDTA and subcultured with a split ratio 1:2. HAoSMC in the experiments were used between passages 2 and 5. Tissues from six donor and nine recipient patients were obtained following informed patient consent and approval from the local hospital ethics committee in accordance with the Declaration of Helsinki.

Analysis of CaSR mRNA expression. Total RNA was isolated from HAoSMC lysates using an RNAqueous-4PCR kit (Ambion) following the manufacturer's protocol. Arterial samples from patients were homogenized in liquid nitrogen and solubilized using the lysis buffer from the same kit. Reverse transcription of total RNA was carried out using the reverse transcription (RT) system with random hexamers (Promega). PCR amplification of CaSR cDNA was performed using the following primers: 5'-TTCCGCAACACACCCATTGTCAAGG-3' and 5'-GGATCCCGTGGAGCCTCCAAGGC-3' (9). PCR reactions (40 µl) were set up using 1x reaction buffer, which contained 16 mM (NH4)2SO4, 67 mM Tris·HCl (pH 8.8), 0.01% Tween 20, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 µM of each primer, and 1 U of BIOTAQ DNA polymerase (Bioline). Amplification was performed using an initial denaturation step (95°C for 5 min) followed by 35 cycles of 95°C (1 min), 58°C (1 min), and 72°C (1 min). In addition, a final elongation step of 72°C for 7 min was included. Control PCR was carried out using primers specific for 18S ribosomal RNA, producing a PCR product of 324 bp. To eliminate amplification of contaminating genomic DNA, as a negative control for RT-PCR, the RT step was omitted. RT-PCR products were separated on a 1% agarose gel. The presence of a 816-bp amplified product was indicative of positive PCR arising from the CaSR-related sequence in cDNA. Purified PCR products were verified by sequencing on an automatic DNA sequencer (ABI, Warrington, UK) using the same primers as for the RT-PCR.

Western blot analysis. HAoSMC were treated with the specified agonists and then harvested. Briefly, cells were washed three times with cold PBS, scraped, and solubilized in 80 µl cold lysis buffer with freshly added protease inhibitor cocktail and phosphatase inhibitor cocktails 1 and 2 (Sigma). Cell debris was pelleted by microcentrifugation at 10,000 g for 10 min at 4°C. Aliquots of cell lysates containing 15 µg protein were separated by SDS-PAGE and Western blotted with the indicated antibodies. Densitometry was performed using Image J Analysis software using blots from three independent experiments with final results normalized using loading controls (smooth muscle {alpha}-actin or total ERK1/2).

Analysis of CaSR protein expression. Tissue samples from patients were homogenized in liquid nitrogen and solubilized on ice using lysis buffer containing 0.25 M Tris·HCl (pH 7.8), 0.5% Igepal, 5 mM DTT, and freshly added protease inhibitor cocktail (Sigma). The lysates were then separated by 7% SDS-PAGE and Western blotted with a polyclonal anti-human CaSR antibody (1:500) (Binding Site) and anti-human smooth muscle {alpha}-actin antibody (Sigma). HKC-8 (human proximal tubule) lysates were used as a positive control for the expression of CaSR protein (1).

Immunocytofluorescence. HAoSMC were allowed to grow to 50% confluence on coverslips pretreated for 5 min with 2% 3-(aminopropyl)triethoxysilane in acetone and then fixed in 4% paraformaldehyde in PBS. Cellular localization of the CaSR was determined as described previously (11). Briefly, nonspecific binding was prevented by blocking for 1 h at 25°C in PBS with 0.01% Triton X-100 containing 10% normal rabbit serum. Cells were incubated with CaSR antibody (1:100) overnight at 4°C. After triple washing with PBS for 5 min and incubation with donkey anti-sheep Alexa Fluor 488-conjugated antibody (1:400) for 1 h at 25°C, coverslips were mounted in Vectashield with 4,6-diamidino-2-phenylindole (Vector Labs) on glass slides. The cells were examined under an oil-immersion objective (x63) using a Leica DMRE laser-scanning confocal microscope with a TCS SP2 scan head. Control coverslips were stained with either secondary antibody alone or primary antibody preabsorbed with a 50-fold excess of immunizing peptide (Alta Biosciences).

Immunohistochemistry. Immunohistochemical staining for the CaSR was performed using a modified version of the protocol described previously (36). Paraffin sections were boiled in 0.01 M sodium citrate buffer (pH 6.0) in a pressure cooker for 90 s. Slides were then incubated in 3% hydrogen peroxide in methanol for 15 min to quench endogenous peroxidase activity and washed in PBS. The sections were incubated with 10% blocking serum followed by anti-human CaSR antibody (1:100) for 30 min. After a further PBS wash, rabbit anti-sheep IgG peroxidase conjugate (1:100) was added to sections for 40 min. CaSR expression was visualized using DAB substrate solution (Vector Labs) and counterstained with Mayer's hematoxylin. Control sections were stained with either secondary antibody alone or primary antibody preabsorbed with a 50-fold excess of immunizing peptide. Von Kossa staining for calcium deposits was carried out as described earlier (24).

ERK1/2 phosphorylation. HAoSMC were plated at 2 x 105cells/well of a six-well plate and grown overnight in complete medium. The following day, the medium was changed and cells were incubated overnight in DMEM:F-12(1:1) medium (Invitrogen) containing 0.2% BSA. The next day, the cells were rinsed in PBS for 5 min before equilibration for 20 min in experimental buffer containing 20 mM HEPES (pH 7.4), 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, and 5.5 mM glucose (26). HAoSMC were then stimulated with increasing concentrations of Ca2+ (0–5 mM) and CaSR agonists (neomycin and gentamycin, 0–300 µM) for 2–30 min. Where indicated, the cells were pretreated for 5 min with 10 µM PD-98059, a specific MEK1 inhibitor (Calbiochem). After incubation the cells were lysed on ice in RIPA lysis buffer (Upstate), separated by 10% SDS-PAGE, and Western blotted with anti-phospho-ERK1/2 (New England Biolabs). Protein concentration in the samples was measured using a DC Bio-Rad protein assay to ensure equal protein loading. In addition, to confirm that ERK protein levels were not altered by the experimental treatment, the samples were also immunoblotted with anti-total ERK1/2.

Inhibition of CaSR expression by specific siRNA. To examine the functional role of the CaSR in SMC, we used small-interference RNA (siRNA) to knock down the level of CaSR expression (Santa Cruz Biotechnology). The CaSR siRNA is a pool of 3 target-specific 20- to 25-nucleotide siRNAs designed to inhibit gene expression. The transfection of siRNA specifically targeted to the CaSR into HAoSMC was performed using Lipofectamine (Invitogen) according to the manufacturer's protocol, as described previously (11). Briefly, Lipofectamine and siRNAs were diluted into OptiMEM medium (Invitrogen). Diluted Lipofectamine lipids were mixed with diluted siRNAs and incubated for 30 min at room temperature for complex formation. Mixtures were further diluted in OptiMEM and added to each well so that the final concentration of siRNAs was 40 nM. Control siRNA-A and Lipofectamine alone were used as negative controls. The effectiveness of transfection (CaSR knockdown) was monitored by Western blotting.

Statistical analysis. All experiments were performed at least three times, and the results are expressed as means ± SD. Statistical analysis was performed using descriptive statistics, a two-tailed paired t-test and one-way ANOVA followed by Tukey's multiple comparison test. P values <0.05 were considered as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CaSR mRNA is expressed in human vascular SMC. Initial investigations examined whether CaSR mRNA was expressed in primary cultures of HAoSMC. One microgram of total RNA was used for RT-PCR with CaSR-specific primers generating an 816-bp fragment within exon 7 of the CaSR gene. A PCR product of the correct size (816 bp) was obtained (Fig. 1A). Purified PCR products were then sequenced and shown to be identical to the human CaSR sequence. As a positive control, 1 µg of total RNA from HKC-8 cells was used, which produced the band of the same size as HAoSMC, but with the expression level in kidney cells being substantially higher than in HAoSMC (Fig. 1A).


Figure 1
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  		Fig. 1. Calcium-sensing receptor (CaSR) mRNA expression in human artery. A: RT-PCR was carried out using total RNA extracted from human aortic smooth muscle cells (HAoSMC), normal renal arteries from kidney donors, epigastric arteries from patients with renal failure, and primers specific for human CaSR. HKC-8 RNA was used as a positive control. Control PCR was performed using primers specific for 18S ribosomal RNA. B: no PCR products were detected in the absence of reverse transcriptase (RT–) or cDNA (H2O).

 
In addition, RNA isolated from normal renal and epigastric arteries of transplant donors and ESRD recipients undergoing renal transplant was analyzed. Figure 1A shows RT-PCR analysis in six representative donor and recipient patients. It produced cDNA products of the expected size, although the level of expression varied considerably and tended to be lower in ESRD recipients. PCR amplification of 18S ribosomal RNA was run in parallel (Fig. 1A). To exclude the possibility of genomic DNA contamination, we ran RT-PCR after omitting the RT step. As expected, no PCR products were detected in the absence of the reverse transcriptase (Fig. 1B).

CaSR protein is present in human vascular SMC. Protein lysates from transplant donor and recipient patient arteries and primary cultures of HAoSMC were separated using 7% SDS-PAGE and blotted against CaSR antibody. HKC-8, used as a positive control, and HAoSMC lysate produced a band of ~160 kDa, consistent with the mature, full-size CaSR (Fig. 2A). Protein lysates from renal and epigastric arteries of renal transplant patients produced doublets of ~150 and 140 kDa (Fig. 2B). Smooth muscle {alpha}-actin was strongly expressed in all samples except HKC-8 cells and confirmed equal loading of protein lysates. We observed a considerable variation in CaSR protein expression between different patients. Densitometry showed that average CaSR expression in six transplant donor patient arteries was almost threefold higher (P < 0.05) than in nine transplant ESRD recipient arteries (Fig. 2C).


Figure 2
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  		Fig. 2. CaSR protein expression in human artery. A and B: cell extracts were separated by 7% SDS-PAGE and Western blotted with anti-CaSR or anti-{alpha}-smooth muscle actin. Protein lysate from HKC-8 cells was used as a positive control. C: densitometry was performed using Image J Analysis software. Values are means ± SD (n = 3) with protein expression shown as standardized fold-increase/decrease from control (HKC-8).

 
The expression of CaSR in HAoSMC was examined by immunocytofluorescence. The CaSR was distributed throughout the cytoplasm and at the membrane (Fig. 3). Limited staining was detected following preabsorption of CaSR antibody and immunizing peptide (Fig. 3C).


Figure 3
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  		Fig. 3. Immunofluorescent confirmation of CaSR expression in human vascular SMC. HAoSMC cells were grown on coverslips and subjected to immunofluorescence staining with a polyclonal antibody against human CaSR and Alexa Fluor 488 secondary antibody (green). Cell nuclei were stained using the DNA-specific dye DAPI (blue). A: nuclear and CaSR staining (magnification x63). Control cells were stained with either secondary antibody alone (B) or primary antibody preabsorbed with a 50-fold excess of immunizing peptide (Alta Biosciences, Birmingham, UK; C).

 
Immunohistochemical staining for CaSR in human tissue. Immunohistochemical staining of human aorta and renal arteries revealed high expression of CaSR protein in SMC as well as in endothelial cells (Fig. 4). Staining was observed both on the plasma membrane and intracellularly. Similarly, strong CaSR staining was observed in smooth muscle and endothelial layers of epigastric artery taken from a 40-yr-old transplant recipient who was on dialysis for 30 mo (Fig. 5A). Importantly, virtually no immunoreactivity was found in arterial sections incubated with CaSR antibody preabsorbed with the immunizing peptide (Fig. 5B) or with the secondary antibody alone (data not shown), which confirmed specificity of the CaSR staining. Analysis of sequential sections of epigastric artery from a 54-yr-old transplant recipient (72 mo on dialysis) revealed that SMC areas undergoing progressive calcification (visualized by von Kossa staining, Fig. 5D) had considerably lower CaSR expression (Fig. 5C).


Figure 4
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  		Fig. 4. Immunohistochemical staining of human aorta, renal, and intrarenal arteries for CaSR. Paraffin sections were stained using anti-CaSR and counterstained with Mayer's hematoxylin. A and B: human aorta (magnification x40 and x200). C and D: human renal artery (magnification x100 and x200). E and F: human intrarenal artery (magnification x200 and x400).

 

Figure 5
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  		Fig. 5. Immunohistochemical staining of human epigastric artery for CaSR and calcification. A and C: epigastric arterial sections were stained using anti-CaSR and counterstained with Mayer's hematoxylin (magnification x200). Negative controls included omission of primary antibody (not shown) and primary antibody preabsorbed with immunizing peptide (B). Von Kossa staining was used for detection of calcium deposits in the arterial wall (D).

 
These data clearly demonstrate that human vascular SMC express both CaSR mRNA and protein and that the level of its expression varies in ESRD patients.

ERK1/2 phosphorylation in HAoSMC treated with CaSR agonists. To establish whether CaSR agonists activate downstream signaling pathways in HAoSMC, we examined the effects of Ca2+, neomycin, and gentamycin on phosphorylation/activation of ERK1/2. HAoSMC cells were treated with Ca2+ (5 mM) and neomycin and gentamycin (both 300 µM) for up to 30 min.

Treatment with 5 mM Ca2+ produced a significant (2- to 5-fold, P < 0.05) increase in ERK1/2 phosphorylation. The effect was observed after 2-min incubation, reached maximum levels by 5 min, and was still apparent after 30 min (Fig. 6A). Analysis of the dose-response showed that ERK1/2 phosphorylation was upregulated only in cells treated with >3 mM Ca2+ (Fig. 6B). Neomycin treatment resulted in a pronounced upregulation of phospho-ERK1/2 (P < 0.01), which peaked at ~10 min and was sustained after 30-min incubation (Fig. 6C). The response was dose dependent, with the maximal effect observed at 100–300 µM (Fig. 6D). The addition of gentamycin elicited effects very similar to those observed in neomycin-treated cultures (see Fig. 6, E and F). Importantly, pretreatment of HAoSMC with 10 µM PD-98059, an MEK1 inhibitor, almost completely abolished neomycin- and gentamycin-induced ERK1/2 activation, confirming the specific nature of the observed effects. It is necessary to note that the changes in ERK1/2 phosphorylation described were not due to unequal protein loading or altered total ERK1/2 expression, as these were controlled by the protein assay and total ERK1/2 expression. Taken together, our findings indicate that CaSR agonists can induce activation of the MEK1/ERK1/2 pathway in HAoSMC.


Figure 6
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  		Fig. 6. Phospho-ERK1/2 expression in HAoSMC treated with calcium, neomycin, and gentamycin. HAoSMC in 6-well plates were incubated overnight in serum-free medium and treated with CaSR agonists. Cells were incubated with 5 mM Ca2+ (A), 300 µM neomycin (C), and 300 µM gentamycin (E) for 0–30 min. Alternatively, HAoSMC were treated with 1–5 mM Ca2+ for 5 min (B) or with 10–300 µM neomycin (D) and gentamycin (F) for 10 min. Where indicated, the cells were preincubated for 5 min with 10 µM PD-98059. Cell lysates were separated by 10% SDS-PAGE and Western blotted with anti-phospho-ERK1/2 and anti-total ERK1/2. Densitometry was performed using Image J Analysis software. Values are means ± SD (n = 3) with antigen expression shown as standardized fold-increase/decrease from control. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control untreated cells. xP < 0.01 compared with cells treated with 300 µM neomycin/gentamycin.

 
ERK1/2 phosphorylation in HAoSMC transfected with CaSR siRNA. To confirm that CaSR agonist-induced activation of the MEK1/ERK1/2 pathway is indeed mediated via the CaSR, we used receptor-specific siRNA. Western blot analysis confirmed knockdown of the CaSR. Transfection with CaSR siRNA resulted in a 60% decrease (P < 0.05) in CaSR expression in HAoSMC with no change in {alpha}-actin expression (Fig. 7, A and B). No significant changes in CaSR level were observed in cells treated with vehicle alone (Lipofectamine) or transfected with control siRNA-A.


Figure 7
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  		Fig. 7. Effect of small-interference RNA (siRNA) on CaSR expression and ERK1/2 activation in HAoSMC. HAoSMC were left untreated (blank), treated with vehicle (Lipofectamine), or transfected with control siRNA-A and CaSR siRNA as described previously. A: cell lysates were Western blotted with anti-CaSR or anti-{alpha}-smooth muscle actin. C: after transfection with CaSR siRNA, HAoSMC were treated with CaSR agonists (300 µM neomycin/gentamycin for 10 min). Cell lysates were Western blotted with anti-phospho-ERK1/2 and anti-total ERK1/2. B and D: densitometry was performed using Image J Analysis software. Values are means ± SD (n = 3) with protein expression shown as standardized fold-increase/decrease from blank (untreated cells). B: *P < 0.05 compared with blank and xP < 0.05 compared with cells transfected with control siRNA-A. D: *P < 0.05 compared with neomycin-treated cells transfected with control siRNA-A and xP < 0.05 compared with gentamycin-treated cells transfected with control siRNA-A.

 
HAoSMC transfected with control siRNA-A or CaSR siRNA were treated with 300 µM neomycin or gentamycin for 10 min. As in previous experiments, we observed a marked upregulation of phospho-ERK1/2 in cells transfected with control siRNA-A (Fig. 7, C and D). However, in HAoSMC transfected with CaSR siRNA, such a response was significantly reduced (P < 0.05), indicating that in HAoSMC CaSR agonists (neomycin and gentamycin) mediate activation of the MEK1/ERK1/2 pathway via the CaSR.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Cloning and characterization of the CaSR from bovine parathyroid cells provided strong evidence in favor of extracellular Ca2+ as a first messenger (12). Recent findings suggest that the CaSR is present in vascular SMC; however, it is still unclear whether human SMC express the CaSR capable of transducing extracellular Ca2+ signal into the cell interior. We hypothesized that in human SMC calcium can act as a first messenger and via the CaSR regulate intracellular signaling events.

RT-PCR with CaSR-specific primers demonstrated the presence of CaSR mRNA in both primary HAoSMC and human arteries. Western blotting of protein lysates from HAoSMC and HKC-8 produced distinct bands of ~160 kDa, which were consistent with the mature form of the CaSR (32). Lysates from the epigastric arteries of renal transplant patients consistently gave double bands of slightly smaller size, 150 and 140 kDa. It is necessary to mention that the size of CaSR detected by Western blotting varies considerably depending on the tissue and cell type, cellular fraction analyzed (membrane or cytosolic), and degree of posttranslational modification (glycosylation) of CaSR protein. However, it is generally agreed that bands of 130–170 kDa represent a mature, fully glycosylated form of the CaSR (17, 37). Notably, the expression of CaSR mRNA and protein in samples from nine transplant recipients with ESRD tended to be lower than in six donor patients.

Immunohistochemistry confirmed the expression of CaSR protein in human vascular SMC. CaSR immunoreactivity was observed in the SMC layer of both large (aorta and renal artery) and small (epigastric and intrarenal artery) vessels. The CaSR seemed to be present both on the plasma membrane and in the cytosol. Such intracellular staining was not surprising as numerous studies have demonstrated intracellular CaSR localization in other cell types (21, 26, 29, 32). It has been suggested that cytosolic localization of the CaSR may be due to a high rate of receptor synthesis, extensive posttranslational modification, or membrane receptor recycling (7, 37).

To examine a possible functional role of the CaSR expressed in vascular SMC, we studied ERK1/2 phosphorylation/activation in response to CaSR agonists, i.e., Ca2+, neomycin, and gentamycin. It has been shown that neomycin and gentamycin are strongly charged at physiological pH and cannot cross the plasma membrane (22, 32). Therefore, the regulatory effects of these antibiotics on ERK1/2 can only be triggered by stimulating the membrane-bound CaSR. ERK1/2 is a component of an important intracellular signaling pathway from the plasma membrane to the cell nucleus, which is known to be downstream of the CaSR and to play a crucial role in cell cycle regulation. Previous studies demonstrated that extracellular Ca2+ can induce ERK1/2 activation in various cell types, including parathyroid and CaSR-transfected HEK293 cells (14), osteoblasts (13), kidney tubular cells (32), and aortic SMC (26). This study revealed a consistent and significant upregulation of phospho-ERK1/2 in HAoSMC incubated with Ca2+, neomycin, and gentamycin. Importantly, phospho-ERK1/2 upregulation induced by neomycin/gentamycin was almost completely abolished by PD-98059, a specific MEK1 inhibitor, indicating that their effects on ERK1/2 were mediated via the classic MEK1/ERK1/2 pathway.

Analysis of the kinetics of the response revealed that induction of phospho-ERK1/2 varied between Ca2+ or gentamycin/neomycin stimulation. The observed kinetics of ERK1/2 activation was consistent with the results obtained by Tfelt-Hansen et al. (28) in cultured rat cardiomyocytes. They found that calcium-induced ERK1/2 activation peaked at 5–10 min, with a maximal response at 6 mM Ca2+ and higher (28). Similar data were reported by Kifor et al. (14) in experiments with bovine parathyroid and CaSR-transfected HEK293 cells. However, the kinetics of ERK activation and dose response to CaSR agonists can vary between cell types. Using an in vitro rat aortic vascular SMC model, Smajilovic et al. (26) demonstrated strong ERK1/2 activation induced by 3 mM Ca2+ at 15 min, later than ERK1/2 phosphorylation observed in our experiments. In tubular opossum kidney cells, ERK1/2 activation in response to neomycin was more rapid and reached a maximum at 5 min (32). Consequently, the differences in the duration of ERK1/2 activation can be detected by multiple immediate early gene products and, therefore, produce different effects on the cell cycle (16). Inhibition of ERK1/2 activation in HAoSMC with CaSR expression knocked down by transfection with specific siRNA confirmed that the observed neomycin/gentamycin-induced MEK1/ERK1/2 activation was indeed mediated via the CaSR.

Patients with CKD have a number of additional cardiovascular risk factors that may be responsible for the accelerated arterial calcification, such as duration of dialysis and disorders of mineral metabolism. Moe and Chen (15) analyzed calcified inferior epigastric arteries of ESRD patients and found expression of Cbfa1 and several bone-associated proteins in both the intima and medial layers. They also showed that in cultured SMC, the addition of pooled serum from dialysis patients accelerated mineralization and increased expression of the osteoblast markers. These data support growing experimental evidence suggesting a major role for vascular SMC in arterial calcification (25, 30). Our data strongly suggest that human vascular SMC express both CaSR mRNA and protein. Significantly, the observation that there are considerably lower levels of CaSR expression in epigastric arteries of patients with advanced renal impairment compared with healthy transplant donors suggests that CaSR expression tends to decline as CKD progresses. Possibly, the observed decrease in CaSR expression in epigastric arteries of patients with ESRD could be due to SMC transformation into osteoblast-like cells followed by formation of calcium deposits gradually replacing original vascular SMC. Interestingly, we found that in sequential sections of epigastric arteries taken from patients with ESRD (transplant recipients), decreased CaSR expression was accompanied by calcification of SMC areas. A number of factors including elevated circulating calcium level may promote vascular SMC transformation and matrix calcification (20). In addition, Chattopadhyay et al. (8) recently reported that the CaSR stimulated proliferation in rat calvarial osteoblasts. It is possible that stimulation of the extracellular CaSR can accelerate vascular SMC transformation into osteoblast-like cells followed by their proliferation and progressive calcification. However, the precise role of the CaSR in this process remains to be investigated.

In conclusion, we have demonstrated that human vascular SMC express functional CaSR, which, when stimulated, activates the MEK1/ERK1/2 signaling pathway. In ESRD patients, progression of the disease is accompanied by a significant decline in the expression of the CaSR.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by a grant from Diabetes Endocrine and Immersion Trust (R. Bland) and the Coventry Kidney Research Fund and Coventry and Warwickshire Kidney Patient Association. (D. Zehnder).


   ACKNOWLEDGMENTS
 
We are grateful to Professor A. R. Bradwell (Binding Site) for generating CaSR antibody and for an MRC Infrastructure Award (G4500017) "Bioinformatics and Structural Biology in Life Sciences" for CaSR peptide design. Collection of human arteries was enabled by F. T. Lam, Habib Kashi, and Lam Chin Tan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Zehnder, Clinical Sciences Research Institute, Warwick Medical School, Clifford Bridge Rd., Coventry CV2 2DX, UK (e-mail: d.zehnder{at}warwick.ac.uk)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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