A functional single-nucleotide polymorphism in the TRPC6 gene promoter associated with idiopathic pulmonary arterial hypertension

Abstract

Background: Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role in the development of idiopathic pulmonary arterial hypertension (IPAH), whereas a rise in cytosolic Ca2+ concentration triggers PASMC contraction and stimulates PASMC proliferation. Recently, we demonstrated that upregulation of the TRPC6 channel contributes to proliferation of PASMCs isolated from IPAH patients. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC6 gene promoter that are associated with IPAH and have functional significance in regulating TRPC6 activity in PASMCs.

Methods and results: Genomic DNA was isolated from blood samples of 237 normal subjects and 268 IPAH patients. Three biallelic SNPs, -361 (A/T), -254(C/G), and -218 (C/T), were identified in the 2000-bp sequence upstream of the transcriptional start site of TRPC6. Although the allele frequencies of the -361 and -218 SNPs were not different between the groups, the allele frequency of the -254(C-->G) SNP in IPAH patients (12%) was significantly higher than in normal subjects (6%; P<0.01). Genotype data showed that the percentage of -254G/G homozygotes in IPAH patients was 2.85 times that of normal subjects. Moreover, the -254(C-->G) SNP creates a binding sequence for nuclear factor-kappaB. Functional analyses revealed that the -254(C-->G) SNP enhanced nuclear factor-kappaB-mediated promoter activity and stimulated TRPC6 expression in PASMCs. Inhibition of nuclear factor-kappaB activity attenuated TRPC6 expression and decreased agonist-activated Ca2+ influx in PASMCs of IPAH patients harboring the -254G allele.

Conclusions: These results suggest that the -254(C-->G) SNP may predispose individuals to an increased risk of IPAH by linking abnormal TRPC6 transcription to nuclear factor-kappaB, an inflammatory transcription factor.

Figure 1

Identification of 3 SNPs in the 5′-regulatory region of the human TRPC6 gene. A, Schematic diagram of the TRPC6 gene and locations of the amplified PCR segments. B, The location of 3 SNPs, −361(A→T), −254(C→G), and −218(C→T), in the 5′-regulatory region of TRPC6. C, Representative sequence chromatographs for the 3 SNPs in IPAH patients. Arrow denotes position of the SNP. UTR indicates untranslated region.

Figure 2

Nuclear NF-κB proteins of PASMCs specifically bind to the −254(C→G) SNP–generated NF-κB binding site. A, NF-κB activation demonstrated by translocation of p50 and p65 from the cytoplasm to the nucleus. PASMCs from normal subjects were stimulated with (+) or without (−) TNF-α (10 ng/mL). B, Electrophoretic mobility shift assay and competition analysis. TNF-α–stimulated nuclear extracts from PASMCs were incubated with biotin-labeled double-stranded oligonucleotides corresponding to the 24-bp sequences containing the wild-type −254C or mutant −254G probes. Nuclear extract/oligonucleotide complexes (C1 and C2) are indicated by arrows. C, Antibody-mediated supershift assay. NF-κB p50 and p65 antibodies identified complexes C1 as p50/p65 and C2 as p50/p50 NF-κB–containing complexes.

Figure 3

The basal and TNF-α–mediated promoter activities are enhanced in PASMCs transfected with the reporter constructs containing −254G allele. A, Schematic diagram (left) of the reporter constructs used for the promoter assay and the relative promoter activity in control and TNF-α–treated cells (right). The relative β-galactosidase activity (right) represents the mean±SE (plotted on a log scale) of 3 independent experiments performed in quadruplicate. *P<0.05, ***P<0.001 vs control cells. B, COS-7 cells transfected with pBlue(−335/+110) constructs that contain −254C or −254G allele. X-Gal staining was used to detect the β-galactosidase–stained cells. Empty (or promoterless) pBlue TOPO vector was used as control.

Figure 4

Upregulated TRPC6 expression in PASMCs from IPAH patients increases the resting [Ca²⁺]_cyt and enhances agonist-mediated Ca²⁺ influx. A, mRNA and protein expression of TRPC6, determined by RT-PCR (left), Western blot (middle), and immunocyto-chemistry (right), respectively, is significantly higher in IPAH PASMCs than in control PASMCs from NPH patients. B, Representative records of [Ca²⁺]_cyt changes in response to OAG (100 μmol/L) in control (NPH) and IPAH PASMCs. C, Summarized data of the resting [Ca²⁺]_cyt and the amplitude of the OAG-induced increase in [Ca²⁺]_cyt in NPH and IPAH PASMCs. **P<0.01, ***P<0.001 vs NPH PASMCs. D, Representative currents recorded in NPH (left) and IPAH (middle) PASMCs before (Cont) and during (OAG) application with OAG (100 μmol/L). OAG-sensitive currents, generated by subtracting the currents recorded during OAG from the control currents, in NPH and IPAH PASMCs are shown in the right panel.

Figure 5

Downregulation of TRPC6 with siRNA inhibits OAG-induced Ca²⁺ influx in PASMCs from IPAH patients. A, mRNA (left) and protein (right) expression levels of TRPC6, TRPC4, and GADPH are shown in IPAH PASMCs treated with scrambled siRNA, GADPH siRNA, or TRPC6 siRNA. B, Representative records of [Ca²⁺]_cyt changes in response to OAG in IPAH PASMCs treated with scrambled siRNA (left) or TRPC6 siRNA (right). C, Summarized data of the resting [Ca²⁺]_cyt (left) and OAG-induced [Ca²⁺]_cyt increases (right) in IPAH PASMCs treated with scrambled or TRPC6 siRNA. **P<0.01, ***P<0.001 vs scrambled siRNA-treated control cells.

Figure 6

Overexpression of TRPC6 in normal PASMCs (with −254C/C genotype) enhances the OAG-induced increase in [Ca²⁺]_cyt. A, Western blot analysis of TRPC6 in cells transfected with pCMS-EGFP or pCMS-EGFP-hTRPC6^HA. B, Exogenous human TRPC6^HA expression was identified by immunofluorescence staining. Normal PASMCs were transfected with pcDNA3-hTRPC6^HA. HA epitope tag was stained by TRITC-conjugated anti-HA monoclonal antibody. TRPC6 antibody, followed by FITC-conjugated secondary antibody, was used to detect TRPC6 expression. C, Representative records of [Ca²⁺]_cyt changes in response to OAG in PASMCs transfected with pCMS-EGFP or pcMS-EGFP-hTRPC6^HA. D, Summarized data of the resting [Ca²⁺]_cyt and the amplitude of the OAG-induced [Ca²⁺]_cyt increases in cells transfected with pCMS-EGFP or pCMS-EGFP-hTRPC6^HA. ***P<0.001 vs pCMS-EGFP cells.

Figure 7

Overexpression of the inhibitory subunit IκBα attenuates NF-κB–mediated TRPC6 expression and inhibits agonist-mediated Ca²⁺ influx in IPAH-PASMC. A, Overexpression of IκBα (Ad-IκB) inhibits translocation of NF-κB p50 and p65 into the nucleus (left) and diminishes TNF-α–stimulated TRPC6 expression (right). GAPDH, histone (H2A), and p84 antibodies were used as controls for nuclear and cytoplasmic proteins. Experiments were reproduced 3 times. B, Summarized results showing that TNF-α increases the resting [Ca²⁺]_cyt (left) and enhanced OAG-induced [Ca²⁺]_cyt rise (right) in IPAH PASMCs (−254C/G), whereas IκBα (Ad-IκB) abolishes the TNF-α–mediated enhancement of the resting [Ca²⁺]_cyt and OAG-mediated Ca²⁺ influx. C, Summarized results showing that TNF-α negligibly affects the resting [Ca²⁺]_cyt (left) and OAG-induced [Ca²⁺]_cyt increase (right) in normal PASMCs (−254C/C).