The NR4A orphan nuclear receptor NOR1 is induced by platelet-derived growth factor and mediates vascular smooth muscle cell proliferation

Abstract

Members of the nuclear hormone receptor superfamily function as key transcriptional regulators of inflammation and proliferation in cardiovascular diseases. In addition to the ligand-dependent peroxisome proliferator-activated receptors and liver X receptors, this family of transcription factors includes a large number of orphan receptors, and their role in vascular diseases remains to be investigated. The neuron-derived orphan receptor-1 (NOR1) belongs to the ligand-independent NR4A subfamily, which has been implicated in cell proliferation, differentiation, and apoptosis. In this study, we demonstrate NOR1 expression in vascular smooth muscle cells (SMC) of human atherosclerotic lesions. In response to mitogenic stimulation with platelet-derived growth factor (PDGF), SMC rapidly express NOR1 through an ERK-MAPK-dependent signaling pathway. 5'-deletion analysis, site-directed mutagenesis, and transactivation experiments demonstrate that PDGF-induced NOR1 expression is mediated through a cAMP-response element-binding protein (CREB)-dependent transactivation of the NOR1 promoter. Consequently, short interfering RNA-mediated depletion of CREB abolished PDGF-induced NOR1 expression in SMC. Furthermore, PDGF induced Ser-133 phosphorylation of CREB and subsequent binding to the CRE sites of the endogenous NOR1 promoter. Functional analysis demonstrated that PDGF induces NOR1 transactivation of its consensus NGFI-B-response elements (NBRE) in SMC. We finally demonstrate that SMC isolated from NOR1-deficient mice exhibit decreased cell proliferation and characterize cyclin D1 and D2 as NOR1 target genes in SMC. These experiments indicate that PDGF-induced NOR1 transcription in SMC is mediated through CREB-dependent transactivation of the NOR1 promoter and further demonstrate that NOR1 functions as a key transcriptional regulator of SMC proliferation.

FIGURE 1. NOR1 protein is expressed in SMC of human coronary artery atherosclerotic lesions

Paraffin-embedded serial sections of normal human coronary arteries (A) and arteries containing atherosclerotic lesions of different stages (B–E) were stained for smooth muscle α-actin (upper panels) and NOR1 (lower panels). Objective magnifications are ×20 (left panels) and ×100 (right panels).

FIGURE 2. PDGF induces NOR1 expression through ERK/MAPK-dependent signaling pathways

SMC were serum-deprived for 48 h and stimulated with PDGF (25 ng/ml). A, mRNA was isolated at the indicated time points and analyzed for NOR1 expression by Northern blotting. Cohybridization for the housekeeping gene 36B4 was performed as internal control. B, whole cell proteins were isolated at the indicated time points and analyzed for NOR1 protein expression by Western blotting. Cohybridization for GAPDH was performed to assess equal loading. C, quiescent SMC were pretreated with wortmannin (WT, 200 mM), PD98059 (PD, 10 mM), SB203580 (SB, 15 mM), and SP600125 (SP, 25 mM) for 30 min prior to stimulation with PDGF (25 ng/ml). RNA was isolated 2 h after stimulation and analyzed for NOR1 mRNA expression by Northern blotting. The autoradiograms shown are representative of three independently performed experiments using different cell preparations.

FIGURE 3. The proximal NOR1 promoter confers the transcriptional regulation in response to PDGF

A, SMC were transiently transfected with a 4.0-kb NOR1 promoter construct, serum-deprived, and stimulated with vehicle (white bars) or PDGF (25 ng/ml, black bars) for 6 h. B, SMC were transfected with the indicated 5′-deletion series of the 4.0-kb NOR1 promoter. Serum-deprived cells were stimulated with either vehicle (white bars) or PDGF (25 ng/ml, black bars) for 6 h. Following stimulation, cells were harvested, and luciferase activities were analyzed. Transfection efficiency was adjusted by normalizing firefly luciferase activities to Renilla luciferase activities generated by cotransfection with 10 ng of pRL-CMV. Data are presented as mean ± S.E. from three independently performed experiments (*, p < 0.05 versus vehicle). C, schematic illustration of the three cAMP-responsive elements located in the NOR1 promoter at −79, −64, and −53 from the transcription initiation site.

FIGURE 4. PDGF-induced NOR1 expression is mediated through CREB-dependent transactivation of the NOR1 promoter

A, SMC were transiently transfected with 1.7-kb NOR1 promoter constructs bearing mutations of the CRE sites at −79 (Cremt1), −64 (Cremt2), and −53 (Cremt3). B, SMC were cotransfected with the 1.7-kb NOR1 promoter construct and an empty control vector or an expression vector overexpressing a dominant-negative CREB mutant (ACREB). Following transfection, cells were serum-deprived and stimulated with vehicle (white bars) or PDGF (25 ng/ml, black bars) for 6 h. Luciferase activities were analyzed as described in the legend to Fig. 3. Data are presented as mean ± S.E. from three independently performed experiments (*, p < 0.05 versus vehicle). C, SMC were transfected either with scrambled siRNA or CREB siRNA, serum-deprived, and stimulated with PDGF (25 ng/ml) for 6 h. Whole cell lysates were analyzed for NOR1 and total CREB protein expression by Western blotting. Cohybridization for GAPDH was performed to assess equal loading.

FIGURE 5. PDGF stimulates Ser-133 phosphorylation of CREB and binding to the NOR1 promoter through the ERK-MAPK signaling pathway

A, serum-deprived SMC were stimulated with PDGF (25 ng/ml) and analyzed for Ser-133-phosphorylated CREB by Western blotting. Cohybridization for total CREB was employed as control. B, serum-deprived SMC were preincubated with vehicle or PD98059 (10 μmol/liter) for 30 min and stimulated with PDGF (25 ng/ml) for 15 min. Whole cell lysates were analyzed for Ser-133-phosphorylated CREB and phosphorylated p44/42 MAPK. Cohybridization for total CREB and total p44/42 MAPK was employed as control. C, quiescent SMC were stimulated with PDGF (25 ng/ml) for the indicated time points. Following chromatin immunoprecipitation (IP) using a Ser-133-phospho-CREB antibody (4 μg) or control IgG, PCR analysis was performed using primer pairs that cover the CRE sites between −79 and −46 of the NOR1 promoter. Total extract (Input) was used as positive PCR control. D, serum-deprived SMC were preincubated with vehicle or PD98059 (10 μmol/liter) for 30 min prior to stimulation with PDGF (25 ng/ml) for 30 min. Chromatin immunoprecipitation of Ser-133-phosphorylated CREB to the endogenous NOR1 promoter was performed as described in C. All autoradiograms shown are representative of three independently performed experiments. DMSO indicates Me₂SO.

FIGURE 6. PDGF induces NBRE transactivation

SMC were transiently transfected with a reporter construct containing eight copies of the NBRE consensus sequence upstream of a minimal prolactin promoter driving expression of the firefly luciferase gene. Following transfection, cells were serum-deprived and stimulated with PDGF (25 ng/ml) for 12 h. Data are expressed as normalized luciferase activity and presented as mean ± S.E. (*, p < 0.05 versus quiescent cells).

FIGURE 7. NOR1 expression is required for SMC proliferation

Mouse aortic SMC were isolated from littermate wild type mice and NOR1-deficient mice. Equal numbers of cells (0.5 × 10⁵ cells/plate) were plated on 60-mm plates. A, SMC were maintained in DMEM supplemented with 10% FBS. After 2, 4, 6, and 8 days, the cells were harvested, and cell proliferation was analyzed by cell counting using a hemocytometer. Cell proliferation was expressed as fold induction compared with day 0 and presented as mean ± S.E. (*, p < 0.05 versus NOR1^−/−). B, serum-deprived SMC were stimulated with PDGF (25 ng/ml) for the indicated time points, and cell proliferation was analyzed by cell counting. Cell proliferation was expressed as fold induction compared with day 0 and presented as mean ± S.E. (*, p < 0.05 versus NOR1^−/−). C, NOR1 wild type or NOR1-deficient SMC were transfected with eukaryotic expression vectors overexpressing either GFP as control or NOR1. Following transfection, cells were maintained in DMEM supplemented with 20% FBS, and cell proliferation was analyzed after 3 days. Proliferation was expressed as fold induction compared with day 0 and presented as mean ± S.E. (*, p < 0.05 versus day 0).

FIGURE 8. NOR1 stimulates SMC proliferation by regulating cyclin D1 and D2 expression

A, serum-deprived NOR1 wild type and NOR1^−/− SMC were stimulated with PDGF (25 ng/ml) for 6 h, and whole cell lysates were analyzed for NOR1 protein expression by Western blotting. B, serum-deprived NOR1 wild type and NOR1^−/− SMC were stimulated with PDGF (25 ng/ml) for 24 h and analyzed for cyclin D1 and D2 expression by Western blotting. Cohybridization for GAPDH was employed as control to assess for equal loading.