The orphan nuclear receptor ROR alpha is a negative regulator of the inflammatory response

Abstract

Retinoid-related orphan receptor alpha (ROR alpha) (NR1F1) is a member of the nuclear receptor superfamily whose biological functions are largely unknown. Since staggerer mice, which carry a deletion in the ROR alpha gene, suffer from immune abnormalities, we generated an adenovirus encoding ROR alpha1 to investigate its potential role in control of the inflammatory response. We demonstrated that ROR alpha is expressed in human primary smooth-muscle cells and that ectopic expression of ROR alpha1 inhibits TNFalpha-induced IL-6, IL-8 and COX-2 expression in these cells. ROR alpha1 negatively interferes with the NF-kappaB signalling pathway by reducing p65 translocation as demonstrated by western blotting, immunostaining and electrophoretic mobility shift assays. This action of ROR alpha1 on NF-kappaB is associated with the induction of IkappaB alpha, the major inhibitory protein of the NF-kappaB signalling pathway, whose expression was found to be transcriptionally upregulated by ROR alpha1 via a ROR response element in the IkappaB alpha promoter. Taken together, these data identify ROR alpha1 as a potential target in the treatment of chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis.

Fig. 1. RORα is expressed in different vascular SMC types. (A and B) RT–PCR (35 cycles) analysis of RORα and GAPDH mRNA. C-PCR and C-RT are negative controls for PCR and RT, respectively; VSMC, smooth muscle cells from saphenous veins; CASMC, human coronary artery smooth muscle cells; HASMC, human aortic smooth muscle cells. (C) RT–PCR (30 cycles) analysis of RORα mRNA in SMC infected with Ad-RORα1 or Ad-GFP for 24 h. (D) Analysis of RORα protein expression in SMC infected for 24 h with or without Ad-RORα1. Immunocytochemistry experiments were performed as previously described (Chinetti et al., 1998) using a rabbit polyclonal RORα antibody raised against aa 163–225.

Fig. 2. RORα1 inhibits TNFα-induced IL-6, IL-8 secretion and COX-2 expression in SMC. Primary SMC were infected with Ad-RORα1 or Ad-GFP for 16 h and subsequently stimulated for 24 h with TNFα (10 ng/ml). At the end of the treatment period, IL-6 and IL-8 concentrations in the culture medium were determined by ELISAs (R&D systems, UK) (A and B), and COX-2 and RORα protein expression was measured by western blot analysis (C and D). COX-2 protein levels were normalized against cellular β-actin protein content. (NS: non-specific).

Fig. 3. RORα1 inhibits NF-κB activation. (A) PAC1A cells were transfected with the (NF-κB)₃-Luc reporter construct (100 ng) in the presence of pSG5-RORα1 (50 ng) or empty vector (pSG5 50 ng). Two hours after transfection cells were re-fed with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.2% fetal calf serum (FCS) in the presence or absence of LPS (10 µg/ml) for 24 h. (B) Primary SMC were infected with Ad-RORα1 or Ad-GFP for 16 h and subsequently stimulated for 30 min with TNFα (10 ng/ml). Total (T, lower gel) and nuclear (N, upper gel) protein extracts were prepared and subjected to western blot analysis using a p65 antibody (Santa Cruz). (C) Primary SMC were infected with Ad-RORα1 or Ad-GFP for 16 h and subsequently stimulated for 30 min with TNFα (10 ng/ml). Cells were subsequently immunostained using a rabbit polyclonal p65 antibody (Santa Cruz, sc-109).

Fig. 4. RORα1 inhibits NF-κB DNA-binding activities without affecting p65-mediated NF-κB transcriptional activation. (A) Primary SMC were infected with Ad-RORα1 or Ad-GFP for 16 h and subsequently stimulated for 30 min with TNFα (10 ng/ml). Nuclear protein extracts (5 µg) were prepared and subjected to EMSA using an NF-κB and a Sp1 consensus radiolabelled probe (Promega). The shifted complexes are indicated by an arrowhead. A double arrowhead indicates the free probe. (B) PAC1A cells were transfected with the (NF-κB)₃-Luc reporter construct (100 ng) in the presence of pSG5-RORα1 (50 ng) and RSV-p65 (50 ng) or empty vectors (pSG5, 50 ng; RSV, 50 ng).

Fig. 5. RORα1 activates IκBα transcription. (A) Primary SMC were infected with Ad-RORα1 or Ad-GFP for 16 h. Total RNA was prepared and IκBα and GAPDH mRNA levels were measured by northern blot analysis. (B) Aortas from wild-type and Staggerer mice were isolated and total RNA was prepared. IκBα and GAPDH mRNA levels were measured by dot-blot analysis. (C) PAC1A cells were transfected with various IκBα promoter fragments (100 ng) in the presence of pSG5-RORα1 or empty vector (pSG5, 50 ng).

Fig. 6. RORα1, but not RORα2 nor RORα3, activates IκBα transcription and binds to the ROR site within IκBα promoter. (A) PAC1A cells were transfected with the (IκBα-RORE)₂-TK-Luc reporter vector (100 ng) in the presence of RORα1, RORα2 or RORα3 (50 ng), or empty vector (pSG5, 50 ng). (B) PAC1A cells were transfected with the (IκBα-RORE)₂-TK-Luc reporter vector (100 ng) in the presence of RORα1 (50 ng) and RORα1Δ235 or empty vector (50 ng). (C) EMSA was performed using in vitro translated RORα1 protein (Promega) and a radiolabelled IκBα-ROR probe. Increasing concentrations (1×, 10× and 100×) of competitor wild-type (AGGTCA) or mutated (ACCTCA) oligonucleotides were used to demonstrate the specificity of the shifted complex. To supershift the complex, 1 µl of RORα antibody was added to the binding reaction. (S, shifted complex; SS, supershifted complex; FP, free probe).